SPARC-deficient mice have been shown to exhibit impaired glucose tolerance and insulin secretion, but the underlying mechanism remains unknown. Here, we showed that SPARC enhanced the promoting effect of Muscarinic receptor agonist oxotremorine-M on insulin secretion in cultured mouse islets. Overexpression of SPARC down-regulated RGS4, a negative regulator of β-cell M3 muscarinic receptors. Conversely, knockdown of SPARC up-regulated RGS4 in Min6 cells. RGS4 was up-regulated in islets from sparc −/− mice, which correlated with decreased glucose-stimulated insulin secretion (GSIS). Furthermore, inhibition of RGS4 restored GSIS in the islets from sparc −/− mice, and knockdown of RGS4 partially decreased the promoting effect of SPARC on oxotremorine-M-stimulated insulin secretion. Phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 abolished SPARC-induced down-regulation of RGS4. Taken together, our data revealed that SPARC promoted GSIS by inhibiting RGS4 in pancreatic β cells.