Hypoxia-induced mitogenic factor (HIMF), also known as FIZZ1 or RELM, is a profound vasoconstrictor of the pulmonary circulation and a strong mitogenic factor in pulmonary vascular smooth muscle. To further understand the mechanism of these contractile and mitogenic responses, we examined the effect of HIMF on intracellular calcium in human pulmonary smooth muscle cells (HPASMC). Calcium imaging in Fluo-4-loaded HPASMC revealed that recombinant murine HIMF increased intracellular calcium concentration ([Ca²⁺]_i) in a sustained and oscillatory manner. This increase occurred independent of extracellular calcium influx. Pretreatment of HPASMC with U-73122, a specific inhibitor of phosphatidylinositol-phospholipase C (PLC) completely prevented the HIMF-induced calcium signal. The [Ca²⁺]_i increase was also abolished by pretreatment with 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate (IP₃) receptor antagonist. Ryanodine pretreatment did not affect initiation of [Ca²⁺]_i activation or internal release, but reduced the [Ca²⁺]_i levels at the plateau phase. Pretreatment with the G_i-specific inhibitor, pertussis toxin, and the G_s-specific inhibitor, NF-449, did not block the calcium signal. The knockdown of G_q/11 expression did not prevent calcium release but the pattern of calcium release changed from the sustained oscillatory transients with prolonged plateau into a series of short [Ca²⁺]_i transients that return to baseline. However, pretreatment with the tyrosine kinase inhibitor genistein completely inhibited the internal calcium release. These results demonstrate that HIMF can stimulate intracellular calcium release in HPASMC through the PLC signaling pathway in an IP₃- and tyrosine phosphorylation-dependent manner and G_q/11-protein coupled receptor and ryanodine receptor contribute to the increase of [Ca²⁺]_i.