ABBA was reported to be an actin dynamics regulator. However, the molecular mechanism of action of ABBA is still totally obscure. Here, we show that ABBA is ubiquitously expressed in all the examined cultured cells. We found that expression of ABBA in NIH3T3 cells promotes cell spreading. ABBA binds to and markedly promotes cell spreading-induced Rac1 activation. Cell spreading stimulates ABBA activation probably by inducing it tyrosine phosphorylation, which endows ABBA much higher activity to activate Rac1, and attenuates the interaction between ABBA and Rac1. Loss of function suggests that deletion of ABBA in C6-R cells markedly inhibits Rac1 activation and cell spreading; this suggests that and the interaction between ABBA and activated Rac1 is required for ABBA-promoted cell spreading. Taken together, our results indicate that ABBA is activated in response to cell spreading, which markedly promotes cell spreading, and ABBA is required for Rac1 activation and cell spreading. J. Cell. Biochem. 114: 773–781, 2013. © 2012 Wiley Periodicals, Inc.