Vascular smooth muscle cells (SMCs), one of the major cell types of the vascular wall, play a critical role in the process of angiogenesis under both physiological and pathophysiological conditions, including the cancer microenvironment. Previous studies have shown that VEGF-A 165 augments vascular SMC migration via VEGFR2 (KDR/Flk1) pathways. In this study, we found that VEGF-A 165 (recombinant protein or breast tumor cell-secreted) is also capable of inducing migration of VEGFR2-negative human aortic smooth muscle cells (hAOSMCs), and this induction is mediated through a molecular cross-talk of neuropilin-1 (NRP-1), VEGFR1 (Flt-1), and phosphoinositide 3-kinase (PI3K)/Akt signaling kinase. We found that VEGF-A 165 induces hAOSMC migration parallel with the induction of NRP-1 and VEGFR1 expressions and their associations along with the activation of PI3K/Akt. Neutralization of VEGF action by its antibody or inhibition of VEGF-induced PI3K/Akt kinase activation by wortmannin, a PI3K/Akt specific inhibitor, results in inhibition of VEGF-induced hAOSMC migration. Moreover, RNAi-mediated elimination of the NRP-1 expression or blocking of the activity of VEGFR1 by its antibody in hAOSMCs impairs the VEGF-A 165-induced migration of these cells as well as activation of PI3K/Akt kinase. Collectively, these results establish, for the first time, a mechanistic link among VEGF-A 165, NRP-1, VEGFR1, and PI3K/Akt in the regulation of migration of human vascular smooth muscle cells that eventually could be involved in the angiogenic switch.