Exposure of the skin to UVB light results in the formation of DNA photolesions that can give rise to cell death, mutations and onset of carcinogenic events. Specific proteins are activated by UVB and then trigger signal transduction pathways that leads to cellular responses. An alteration of these signaling molecules is thought to be a fundamental event in tumor promotion by UVB irradiation. RhoB GTPase has been identified as a DNA damage inducible gene. RhoB is involved in EGF receptor trafficking, cytoskeletal organization, cell transformation and survival. We have analyzed the regulation of RhoB and elucidated its role in the cellular response of HaCaT keratinocytes to relevant environmental UVB irradiation. We report here that the activated GTP bound form of RhoB is increased rapidly within 5 min of exposure to UVB and then RhoB protein levels increased concomitantly with EGFR activation. Inhibition of UVB-induced EGFR activation prevents RhoB protein expression and AKT phosphorylation, but not the early activation of RhoB. Blocking UVB-induced RhoB expression with specific siRNAs inhibits AKT and GSK3ß phosphorylation, through inhibition of EGFR expression. Moreover, down regulation of RhoB potentiates UVB-induced cell apoptosis. In contrast, RhoB overexpression protects keratinocytes against UVB-induced apoptosis. These results indicate that RhoB is regulated upon UVB exposure by a two-step process consisting of an early EGFR-independent RhoB activation followed by an EGFR-dependent induction of RhoB expression. Moreover, we have demonstrated that RhoB is essential in regulating keratinocyte cell survival after UVB exposure, suggesting its potential role in photocarcinogenesis.