Ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) is one of the deubiquitinating enzymes in the ubiquitin-proteasome system. It has been shown that UCH-L1 could markedly decrease neointima formation through suppressing vascular smooth muscle cell (VSMC) proliferation in the balloon-injured rat carotid. However, whether UCH-L1 plays roles in VSMC migration remains to be determined. In this study, the primary VSMCs were isolated from aortic media of rats and TNF-α to was used to induce VSMC migration. Using a modified Boyden chamber and wound healing assay, it was found that TNF-α can dose and time-dependently induce VSMC migration with a maximal effect at 10 ng/mL. Moreover, UCH-L1 expression increased gradually with the prolonged induction time at 10 ng/mL of TNF-α. UCH-L1 content in VSMC was then modulated by recombinant adenoviruses expressing UCH-L1 or RNA interference to evaluate its roles in cell migration. The results showed that over-expression of UCH-L1 attenuated VSMC migration, while knockdown of it enhanced cell migration significantly no matter whether TNF-α treatment or not. Finally, the effect of UCH-L1 on NF-κB activation was demonstrated by NF-κB nuclear translocation and DNA binding activity, and the levels of IL-6 and IL-8 in cell culture media were examined by ELISA. It was showed that UCH-L1 over-expression inhibited NF-κB activation and decrease IL-6 and IL-8 levels, while knockdown of it enhanced NF-κB activation and increase IL-6 and IL-8 levels during TNF-α treatment. These data suggest that UCH-L1 can inhibit TNF-α-induced VSMCs migration, and this kind of effect may partially due to its suppression role in NF-κB activation.