Transient receptor potential-3 (TRPC3) channels play a predominant role in forming non-selective cation channels (NSCCs) in airway smooth muscle cells (ASMCs), and are significantly increased in their activity and expression in asthmatic ASMCs. To extend these novel findings, we have explored the regulatory mechanisms that control the activity of TRPC3 channels. Our data for the first time reveal that inositol 1,4,5-trisphosphate (IP3), an important endogenous signaling molecule, can significantly enhance the activity of single NSCCs in ASMCs. The analog of diacylglycerol (another endogenous signaling molecule) 1-oleyl-2-acetyl-sn-glycerol (OAG), 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) and 1-stearoyl-2-linoleoyl-sn-glycerol (SLG) all augments NSCC activity. The effects of IP₃ and OAG are fully abolished by lentiviral shRNA-mediated TRPC3 channel knockdown (KD). The stimulatory effect of IP₃ is eliminated by heparin, an IP₃ receptor (IP₃R) antagonist that blocks the IP₃-binding site, but not by xestospongin-C, the IP3R antagonist that has no effect on the IP₃-binding site. Lentiviral shRNA-mediated KD of IP3R1, IP₃R2 or IP₃R3 does not alter the excitatory effect of IP₃. TRPC3 channel KD greatly inhibits IP₃ -induced increase in [Ca²⁺]_i. IP3R1 KD produces a similar inhibitory effect. TRPC3 channel and IP₃R1 KD both diminish the muscarinic receptor agonist methacholine-evoked Ca²⁺ responses. Taken together, we conclude that IP₃, the important intracellular second messenger, may activate TRPC₃ channels to cause extracellular Ca²⁺ influx, in addition to opening IP₃Rs to induce intracellular Ca²⁺ release. This novel extracellular Ca²⁺ entry route may play a significant role in mediating IP₃-mediated numerous cellular responses in ASMCs and other cells.