Berlin Mayor Klaus Wowereit, a Social Democrat, was forced to abstain from the EU treaty vote because of dissent from his junior partners in the local parliament, the hard-line socialist Left party, Die Linke.
The Left has rejected the treaty, a new legal framework for the EU that replaces the rejected European constitution, as socially irresponsible.
Wowereit said before the vote that the Left had shown "they are not capable of governing."
The Berlin mayor said it would be difficult to continue to work together if the Left continues to take its marching orders from national party co-chairman Oskar Lafontaine, himself a refugee from Wowereit's more moderate Social Democratic Party.
Berlin's was the only abstention on the EU treaty, already approved with a majority of nearly 90 percent in the lower house of the German parliament, the Bundestag, on April 24. Left party MPs opposed the treaty in that vote as well.
The upper house of parliament, the Bundesrat, represents Germany's 16 federal states.
Signed in December in Lisbon, the EU treaty has since been ratified by 13 of the 27 EU member countries and is planned to take effect on Jan. 1, 2009.
The Lisbon treaty amends existing EU treaties and replaces the controversial European constitution rejected by French and Dutch voters in 2005. It includes most of the provisions in the failed constitution.