The main sterol of the human cell membrane is cholesterol, whereas in yeast it is ergosterol. In this study, we constructed a cholesterol-producing yeast strain by disrupting the genes related to ergosterol synthesis and inserting the genes related to cholesterol synthesis. The total sterols of the mutant yeast were extracted and the sterol composition was analyzed by GC-MS. We confirmed that cholesterol was produced instead of ergosterol in yeast and subsequently examined the activity of the yeast G-protein-coupled receptor (GPCR) Ste2p. Ste2p signaling was assessed in wild type (WT) with ergosterol and the cholesterol-producing yeast instead of ergosterol to determine whether sterol composition affects the activity of the yeast GPCR. Our results demonstrated that Ste2p could transduce a signal even in the cholesterol-rich membrane, but the maximum signal intensity was weaker than that transduced in the ergosterol-rich original (WT) membrane. This result indicates that sterol composition affects the activity of yeast GPCRs, and thus, this provides new insight into GPCR-mediated transduction using yeast for future fundamental and applied studies on GPCRs from yeast to other organisms.