Since Ca-sensing receptor (CaSR) in human vascular smooth muscle are largely unknown, we sought to study the activation of CaSR via Ca²⁺ signaling in human aortic smooth muscle cells (HASMC). Superfusion of Ca²⁺ (0.2-2 mM) raised cytoplasmic free Ca²⁺ concentrations ([Ca²⁺]_cyt) in HASMC in a dose-dependent manner; however, thapsigargin (1 mM), ionomycin (10 mM), or caffeine (10 mM) could not induce detectable [Ca²⁺]_cyt. Ca²⁺-induced [Ca²⁺]_cyt increase was significantly inhibited by PTX (100 ng/ml), U73122 (1 mM), chelerythrine (10 mM); but was potentiated by PMA (1 mM). Blockade of TRPC channels with 2-APB (100 mM), SK&F69365 (50 mM) or La3+ (10 mM) significantly inhibited CaSR-mediated [Ca²⁺]_cyt, but activation of TRPC6 channels with flufenamic acid (FFA, 100 mM) potentiated CaSR-mediated [Ca²⁺]_cyt. KB-R7943 (10 mM) and SN-6 (10 mM), two selective inhibitors for the reverse mode of Na⁺/Ca²⁺ exchanger (NCX), significantly inhibited CaSR-mediated [Ca²⁺]_cyt signal in HASMC. Finally, protein expression of NCX1, TRPC6 and PKCa has been detected in HASMC. Taken together, our data suggest that activation of CaSR mediates Ca²⁺ entry through a functional coupling of TRPC6 and the reverse mode of NCX1 in HASMC, which may be regulated by G protein, PLC and PKCa.