ABSTRACTCell division control protein 42 (CDC42) homolog is a small Rho GTPase enzyme that participates in such processes as cell cycle progression, migration, polarity, adhesion, and transcription. Recent studies suggest that CDC42 is a potent tumor suppressor in different tissues and is related to aging processes. Although DNA damage is crucial in aging, a potential role for CDC42 in genotoxic stress remains to be explored. Migration, survival/proliferation and DNA damage/repair experiments were performed to demonstrate CDC42 involvement in the recovery of HeLa cells exposed to ultraviolet radiation‐induced stress. Sub‐lines of HeLa cells ectopically expressing the constitutively active CDC42‐V12 mutant were generated to examine whether different CDC42‐GTP backgrounds might reflect different sensitivities to UV radiation. Our results show that CDC42 constitutive activation does not interfere with HeLa cell migration after UV radiation. However, the minor DNA damage exhibited by the CDC42‐V12 mutant exposed to UV radiation most likely results in cell cycle arrest at the G2/M checkpoint and reduced proliferation and survival. HeLa cells and Mock clones, which express endogenous wild‐type CDC42 and show normal activity, are more resistant to UV radiation. None of these effects are altered by pharmacological CDC42 inhibition. Finally, the phosphorylation status of the DNA damage response proteins γ‐H2AX and p‐Chk1 was found to be delayed and attenuated, respectively, in CDC42‐V12 clones. In conclusion, the sensitivity of HeLa cells to ultraviolet radiation increases with CDC42 over‐activation due to inadequate DNA repair signaling, culminating in G2/M cell accumulation, which is translated into reduced cellular proliferation and survival. J. Cell. Biochem. 116: 2086–2097, 2015. © 2015 Wiley Periodicals, Inc.