tNOX, a tumor-associated NADH oxidase, is a growth-related protein present in transformed cells. In this study, we employed RNA interference (RNAi)-mediated down-regulation of tNOX protein expression to explore the role of tNOX in regulating cell growth in human cervical adenocarcinoma (HeLa) cells. In this first reported use of RNAi to decrease tNOX expression, we found that HeLa cell growth was significantly inhibited by shRNA-knockdown of tNOX. Furthermore, cell migration and membrane association of Rac were decreased concomitantly with the reduction in tNOX protein expression. These results indicate that shRNA targeting of tNOX inhibits the growth of cervical cancer cells, and reduces cell migration via a decrease in the membrane association of Rac. We propose that tNOX is a potential upstream mediator of Rho activation that plays a role in regulating cell proliferation, migration, and invasion.