Spurs' second string failed to deliver the performance ailing manager Harry Redknapp would have wanted as they were comfortably beaten by their Russian opponents, who now go top of the group.
Sebastien Bassong needlessly gave away a free-kick on the edge of the box 10 minutes after half-time and Bebras Natcho took full advantage by clipping the set-piece over the wall and past Carlo Cudicini.
Had it not been for a number of excellent saves from Cudicini, the scoreline could have been much worse and Spurs did not register a shot on target throughout the match.
Despite having internationals William Gallas, Jermain Defoe and Roman Pavlyuchenko among their ranks, Spurs were sloppy throughout and even the most simple of tasks eluded them at times, in contrast to their previous strong performances in the competition.
With a home game against PAOK Salonika and trip to Dublin to face Shamrock Rovers coming up, Redknapp still should be confident of leading his team through to the last 32 when he returns to action following his lay-off.
Redknapp was forced to miss the game after having a heart procedure, but the Tottenham boss was believed to be watching at home.
That meant the likes of Gallas, Defoe and Steven Pienaar had a chance to impress the 64-year-old and earn a place in the team for Sunday's Barclays Premier League game at Fulham.
Youngsters Tom Carroll, Andros Townsend and Ryan Fredericks were also drafted into a team that contained none of the XI who played in Sunday's win against QPR while former Newcastle striker Obafemi Martins started up front for Rubin.
The game started on a bizarre note, with a Rubin fan running onto the pitch in the second minute on what was a predictably cold night in a city more than 450 miles east of Moscow.
Jake Livermore grabbed the fan by his chest and he fell to the floor before the intruder was dragged off unceremoniously by four stewards.
Pavlyuchenko, no doubt keen to make an impression in his homeland, ventured forward but blasted his shot five yards wide.
Rubin had the first real chance of the game in the 24th minute when Alan Kasaev got on the end of Gokdeniz Karadeniz's cross but his shot flew a foot over Cudicini's goal.
The home crowd sensed Tottenham's nerves and the home team grew in confidence. Cudicini pulled off the first of a string of good saves when he tipped the winger's shot wide after coming to punch out a cross.
The former Chelsea stopper then defied his age to dive athletically and tip Natcho's effort wide just before the half-hour.
Cudicini was called upon again from the resulting corner to block Roman Sharonov from 10 yards as Rubin kept piling on the pressure.
Sharonov bodychecked Pavlyuchenko on the edge of the Rubin box, but referee Florian Meyer waved play on, allowing Aleksandr Ryazantsev to race up the other end before drawing another top-drawer save from Cudicini at his near post.
Spurs were struggling to get into the game, but they suddenly sparked into life six minutes before half-time when Defoe held off Salvatore Bocchetti's challenge to square for Pavlyuchenko, but the sliding Russian timed his run a second too late.
Fredericks advanced down the right and shot into the side-netting to give Spurs a strong start to the second half but they were soon undone by Bassong's mistake.
The centre-half, who admitted today that he was looking to leave the club in January, hacked down Ryazantsev after losing the ball while trying to dribble the ball to safety from inside his own box.
The Cameroon defender was booked for the tackle and Defoe also entered the book for protesting the decision. Spurs paid dearly for the mistake as Natcho whipped a dipping shot over the wall and into the bottom right-hand corner of Cudicini's goal to give the home side the lead with 10 minutes of the second half gone.
Spurred on by the goal, Karadeniz then pushed forward and broke into the Spurs box, but Gallas put in a crucial intervention to hook his cross clear.
Karadeniz and Natcho then combined to send Ryazantsev through but Cudicini came out to push the Russian's shot over. Bocchetti volleyed just over from a Natcho corner as the Rubin pressure showed no sign of relenting.
Gallas came off for 19-year-old Dean Parrett with 20 minutes left and Harry Kane replaced a disappointing Pavlyuchenko.
Cudicini then saved well with his legs from Karadeniz with 11 minutes left before the 38-year tipped Valdez's effort wide with another top-class save.
It was Rubin who looked the more likely to score in added time, with Cudicini pulling off another two saves before the final whistle brought to an end a disappointing night for the Londoners.