ARHGAP21 is highly expressed in the heart, which demonstrates activity over Cdc42 and interacts with proteins of the cytoskeleton and adherent junctions. The main cause of cardiac hypertrophy is mechanical stimulus; therefore we analyzed ARHGAP21 expression after acute mechanical stress in the myocardium and its association with FAK and PKCζ. We demonstrated that ARHGAP21 is relocated to Z-lines and costameres after pressure overload, and interacts with PKCζ and FAK in control rats (sham), rats submitted to aortic clamping and spontaneously hypertensive rats (SHR). Co-transfection using ARHGAP21 and PKCζ constructions demonstrated that ARHGAP21 associates with PKCζ-GST and endogenous FAK. Pulldown assay showed that ARHGAP21 binds to the C-terminal region of FAK. Moreover, ARHGAP21 binds to PKCζ phosphorylated on Thr410 in sham and SHR. However, ARHGAP21 only binds to FAK phosphorylated on Tyr925 of SHR. Additionally, PKCζ is phosphorylated by mechanical stimuli. 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