Prolonged activation and proliferation of hepatic stellate cells (HSCs) usually results in the initiation and progression of liver fibrosis following injury. Recent studies have shown that Substance P (SP) participates in the development of fibrosis. However, whether SP is involved in liver fibrosis, especially in the activation and proliferation of HSCs, is largely unknown. In the present study, we measured the effects of a series of concentrations of SP on the cell viability and activation of HSC-T6 cells and LX2 cells. The underlying mechanism was also investigated. We found that SP effectively increased cell viability, both in an MTT assay (p < 0.05) and in a lactate dehydrogenase activity assay (LDH) (p < 0.05). Moreover, SP upregulated the protein expression of α-SMA and Collagen I (both p < 0.05) and decreased the release of lipid droplets (LDs) (p < 0.05), all of which are associated with HSC activation. Apoptosis analysis revealed that SP can attenuate the increase of cell apoptosis induced by serum withdrawal (p < 0.05). Furthermore, these effects were all blocked by an SP receptor antagonist, L732138. More importantly, L732138 decreased the activation of the TGF-β1/Smad3 signaling pathway, which is highly associated with liver fibrosis. Taken together, our results demonstrate that SP can promote HSC proliferation and induce HSC activation via the TGF-β1/Smad3 signaling pathway.