The 30-year-old, who can operate at left-back or centre-back, has signed a four-year deal with the option of a further year.
There is also a £40million buy-out clause in the Frenchman's contract.
Six foot four inch Mathieu started his career with Sochaux, before joining Toulouse and then Valencia in 2009, going on to make 177 appearances for the La Liga side.
Two of Mathieu's four goals last season came against Real Madrid.
Also a full international, albeit with only two caps to date, Mathieu's signing is seen by many in Spain to be filling the gap in Barcelona's defence left by the retired Carlos Puyol.