As a special member of the Rho family, RhoB exhibits distinct biological activities and plays a unique role in tumorigenesis, and its protein levels are tightly regulated compared with other family members. Acting as a tumor suppressor, RhoB has attracted increasing attention in targeted cancer therapy. However, RhoB has been reported to promote development of certain types of tumors, in which the underlying molecular mechanisms remain unclear. In this article we summarize the studies on post-translational modifications of RhoB, particularly its ubiquitination and sumoylation, and their roles in determining the fate of tumor cells. Under normal physiological conditions, RhoB is targeted to ubiquitination and degradation by E3 ubiquitin ligase Smurf1, therefore maintaining relatively low protein levels of RhoB for cell survival. In response to single strand DNA damage, activated Chk1 phosphorylates Smurf1 to induce its self-degradation, resulting an accumulation of RhoB. Meanwhile, RhoB is also phosphorylated by Chk1, leading to dissociation of RhoB from plasma membrane. Phosphorylated RhoB is further sumoylated by SUMO E3 PIAS1 in cytosol, which is required for translocation of TSC2 to lysosomes to inhibit mTORC1 activity and subsequent initiation of autophagy. Hence, RhoB plays a pivotal role in determining cell fate by switching between its ubiquitination and sumoylation. These multiple layers of regulation enable RhoB to execute appropriate cellular responses in different cellular states and environmental conditions. Further exploring the regulatory mechanisms of RhoB in tumor development may provide valuable clues and ideas for cancer treatment by targeting RhoB.