The French defender, who turns 31 in October, had been involved in a drawn-out tug of war with Valencia over his desire to move to the Camp Nou.
He has now moved to Barca on a four-year deal, which includes an option for a further year.
"Valencia CF thanks Mathieu for his professionalism and performance in the five seasons he has played in our club," read a statement.
Mathieu's buyout clause is set at €50m and the former Toulouse man will help fill the void in defence left by the retirement of long-serving Barcelona captain Carles Puyol.
While Mathieu can be utilised centrally, he is also able to play as a left-back.
New coach Luis Enrique had already strengthened Barca's attacking ranks with the addition of Luis Suarez, while also bringing in Ivan Rakitic of Sevilla.
Barca have also signed two goalkeepers in the form of Marc-Andre ter Stegen and Claudio Bravo.