Receptor for activated protein kinase C1 (RACK1) is an intracellular scaffolding protein known to interact with the inositol-1,4,5-trisphosphate receptor and thereby enhance calcium release from the sarcoplasmic reticulum. Because calcium signaling may affect vascular smooth muscle cell proliferation, we investigated whether RACK1 regulates proliferation of rat preglomerular microvascular smooth muscle cells. Western blot analysis indicated that preglomerular microvascular smooth muscle cells robustly express RACK1 protein, and coimmunoprecipitation experiments demonstrated that RACK1 binds the inositol-1,4,5-trisphosphate receptor. RACK1 small interfering RNA (siRNA) decreased RACK1 mRNA and protein expression, significantly (P=0.0225) reduced steady-state basal levels of intracellular calcium (6712±156 versus 7408±248, arbitrary fluorescence units in RACK1 siRNA-treated versus control cells, respectively) and significantly (P<0.0001) decreased cell proliferation by ≈50%. Xestospongin C and 2-aminoethoxydiphenyl borate (antagonists of inositol-1,4,5-trisphosphate receptors), cyclopiazonic acid (sarcoplasmic reticulum Ca(2+)-ATPase inhibitor), and calmidazolium (calmodulin inhibitor) mimicked the effects of RACK1 siRNA on proliferation, and RACK1 siRNA had no additional effects on proliferation in the presence of these agents. RACK1 siRNA did not affect the expression of cyclin D1/2 or phosphorylation of retinoblastoma protein (progrowth cell cycle regulators), yet it caused compensatory decreases in the expression of p21(Cip1/Waf1) and p27(Kip1) (antigrowth cell cycle regulators). Like preglomerular microvascular smooth muscle cells, glomerular mesangial cells also expressed high levels of RACK1, and RACK1 siRNA inhibited their proliferation. In conclusion, RACK1 modulates proliferation of preglomerular microvascular smooth muscle cells and glomerular mesangial cells, likely via the inositol-1,4,5-trisphosphate receptor/calcium/calmodulin pathway. RACK1 may represent a novel druggable target for treating renal diseases, such as glomerulosclerosis.