Regulation of nestin gene expression is largely unknown despite that it is widely used as a progenitor cell marker. In this study, we showed that nestin expression is regulated by the thrombin-mediated EGFR transactivation in serum-deprived primary cultures of rat vascular smooth muscle cells (VSMCs). This resulted from the direct binding of thrombin to PAR-1 rather than indirectly affecting through the binding to thrombomodulin, as demonstrated by thrombomodulin RNAi. In this process, the PAR-1-induced c-Src plays a critical role through two routes; one was the direct intracellular phosphorylation of EGFR and the other was the extracellular activation of the MMP-2-mediated shedding of HB-EGF. The transactivated EGFR then led to the downstream Ras–Raf–ERK signaling axis, but not the p38 or JNK pathways. In addition, the EMSA experiment showed that the transcriptional factor Sp1 is critical for the thrombin-induced nestin expression in rat VSMCs. Furthermore, RNAi of nestin attenuated the thrombin-induced cell proliferation, indicating that thrombin-induced nestin expression and cell proliferation share the same EGFR transactivation mechanism. This study also suggested that nestin may play an important role in cell proliferation induced by the thrombin-mediated EGFR transactivation.