Deletion of the short arm of chromosome 3 is one of the most frequent genetic alterations in many solid tumors including nasopharyngeal carcinoma (NPC), suggesting the existence of one or more tumor suppressor genes (TSGs) within the frequently deleted region. A putative TSG RBMS3 (RNA binding motif, single stranded interacting protein 3), located at 3p24-p23, has been identified in our previous study. Here, we reported that downregulation of RBMS3 was detected in 3/3 NPC cell lines and 13/15 (86.7%) primary NPC tissues. Functional studies using both overexpression and suppression systems demonstrated that RBMS3 has a strong tumor suppressive role in NPC. The tumor suppressive mechanism of RBMS3 was associated with its role in cell cycle arrest at the G1/S checkpoint by upregulating p53 and p21, downregulating cyclin E and CDK2, and the subsequent inhibition of Rb-ser780. Further analysis demonstrated that RBMS3 had a pro-apoptotic role in a mitochondrial-dependent manner via activation of caspase-9 and PARP. Finally, RBMS3 inhibited microvessel formation, which may be mediated by down-regulation of MMP2 and β-catenin and inactivation of its downstream targets, including cyclin-D1, c-Myc, MMP7, and MMP9. Taken together, our findings define a function for RBMS3 as an important tumor suppressor gene in NPC.