Objective: To observe the inhibitory effect of rapamycin PLGA nanoparticles (RAPA-PLGA-NPs) on proliferation of vascular smooth muscle cells (VSMCs). Methods: Rat VSMCs were prepared by primary culture and identified by immunohistochemical staining for α-actin. Cell uptake of RAPA-PLGA-NPs was observed by using transmission electron microscopy (TEM). The effects of RAPA-PLGA-NPs on the growth and expression of the proteins downstream to mTOR (mammalian target of rapamycin) of VSMCs were determined by MTT assay and Western blot analysis, respectively. Meanwhile, the free rapamycin served as comparison. Results: Rat VSMCs were successfully cultured and identified. TEM results showed significant amount of RAPA-PLGA-NPs uptake into the cytoplasm of VSMCs. The MTT results showed that RAPA-PLGA-NPs (1, 10 and 100 ng/mL) inhibited the proliferation of VSMCs in a concentration dependent manner (all P<0.05), and the inhibitory effect of RAPA-PLGA-NPs at 1 ng/mL was similar to that of the free rapamycin at 10 ng/mL (P>0.05). Western blot results showed that RAPA-PLGA-NPs of both concentrations (1 and 10 ng/mL) markedly reduced phosphorylation levels of S6K1 and 4E-BP1 of VSMCs, and both effects were more obvious than that of the free rapamycin (10 ng/mL). Conclusion: RAPA-PLGA-NPs are of enhanced permeability, can be rapidly taken up by cells, and have better biological effects than their free forms.