RhoBTB proteins are atypical members of the Rho family of small GTPases. Two of the three RhoBTB proteins, RhoBTB1 and RhoBTB2, have been proposed as tumor suppressors and might function as adaptors of Cul3-dependent ubiquitin ligase complexes. Using yeast two-hybrid analysis and co-immunoprecipitation we show that all three RhoBTB proteins interact with Cul3. The interaction requires the N-terminal region of Cul3 and the first BTB domain of RhoBTB. RhoBTB3, the only RhoBTB with a prenylation motif, associates with vesicles that are frequently found in the vicinity of microtubules, suggesting a participation in some aspects of vesicle trafficking. We also show that RhoBTB2 and RhoBTB3 are capable of homo and heterodimerizing through the BTB domain region. The GTPase domain, which does not bind GTP, is able to interact with the BTB domain region, thus preventing proteasomal degradation of RhoBTB. This fits into a model in which an intramolecular interaction maintains RhoBTB in an inactive state, preventing the formation or the functionality of Cul3-dependent complexes. We also report a significantly decreased expression of RHOBTB and CUL3 genes in kidney and breast tumor samples and a very good correlation in the expression changes between RHOBTB and CUL3 that suggests that these genes are subject to a common inactivation mechanism in tumors.