However, it is thought to be a symbolic gesture as he has been named last on the candidate list. Barring a landslide, he will not gain office.
He is an icon of the city having won 14 titles in four years at the club.
Last year he returned to Barcelona for a non-binding referendum in which 80% of voters backed leaving Spain.
Now managing Bayern Munich in Germany, Mr Guardiola is standing for a coalition of two pro-independence parties.
The Convergencia Democratica de Catalunya and Esquerra Republicana de Catalunya parties are running a joint list of candidates.
Before becoming Barcelona manager in 2008, he was the club's captain and was schooled at its famed La Masia youth academy.
He also played for the Catalan national team - an unofficial squad that plays friendly matches.
His former club have historically been seen as a standard bearer for Catalan identity. Chants supporting an independent Catalonia frequently ring out around their Camp Nou stadium.
Ahead of last November's referendum, the former midfielder signed an open letter appealing for the result to be respected.
"We Catalans, along with our democratic government and institutions, want to vote so we can decide what kind of relationship we want to establish with Spain for the future," he wrote alongside other high-profile Catalans.
Catalonia is a wealthy region of 7.5 million people and contributes more to the Spanish economy than it gets back through central government funds.
Economic and cultural grievances have fuelled Catalan nationalism.
Former Barcelona football manager Pep Guardiola will run for office as a pro-Catalan independence candidate in September's regional elections.