A costly error from goalkeeper Allan McGregor just before the hour mark allowed Hans Vanaken to record the only goal of the game and leave Steve Bruce’s side in need of a positive result at the KC Stadium in the return date next Thursday.
Only McGregor and James Chester kept their places from Hull's Premier League opener against Queens Park Rangers at the weekend in a much-changed side.
Yannick Sagbo had the best chance of the first half for the visitors in the seventh minute and should have given Hull the lead, but he scuffed his shot allowing the Lokeren defence to clear.
David Meyler and Robbie Brady both tried their luck from distance but Lokeren goalkeeper Davino Verhulst easily gathered their efforts before Sone Aluko put a chance over the bar.
McGregor was called into action at the other end to make a couple of saves and watched as Sergio Dutra Junior put a chance over the top before Meyler fired wide from 20 yards as the first half ended deadlocked at 0-0.
There were no changes to either side at the start of the second period, with McGregor quickly brought into the action after saving a chance by midfielder Nill De Pauw.
The home side did take the lead soon after, though, when Vanaken scored from close range in the 58th minute after the Hull keeper was at fault.
McGregor took too much time on the ball and gifted possession to Vanaken, who rounded the Tigers' stopper and rolled the ball into an empty net.
Aluko tried his luck at the other end, but dragged his low effort just wide of the post from about 25 yards out, before Dutra again skied one over the bar on the turn.
In the 63rd minute, Lokeren defender Giorgos Galitsios chanced his arm with a long-range strike which drifted wide of the Hull goal before McGregor was called upon to make a crucial save to deny De Pauw.
Hull boss Bruce responded with 20 minutes remaining by bringing on summer signing Tom Ince and striker Nikica Jelavic in a bid to get back on terms ahead of the return leg.
Sagbo and Brady made way and Ince came close to making an almost immediate impact when his goal-bound header was diverted behind by Verhulst with just over 10 minute remaining.
Ince had calls for a penalty waved away by the referee shortly afterwards and Chester went agonisingly close to making it 1-1 as the clock ticked down, but he missed the target with a free header from Aluko's cross.
Hull were unable to find their cutting edge in the time that remained, meaning the Belgian side will take a one goal advantage into the second leg.