Because we recently identified the Golf subunit in rat pancreatic -cells, we investigated the downstream effectors and the biological functions of this G protein in HEK-293T cells and the insulin-secreting mouse TC-3 cell line. With the use of transient transfection of HEK-293T cells with constitutively activated Golf (GolfQ214L, i.e., AGolf), together with expression vectors encoding the adenylyl cyclase (AC) isoforms (AC-I to -VIII and soluble AC), compared with cotransfections using AGs (GsR201C), we observed that AGolf preferentially activates AC-I and -VIII, which are also expressed in -cells. Stable overexpression of wild-type or AGolf in TC-3 cells resulted in partial attenuation of insulin secretion and biosynthesis, suggesting that chronic activation of the Golf-signaling pathway is associated with -cell desensitization. In agreement, transfected TC-3 cells present a decreased insulin content with respect to parental cells, whereas the proinsulin convertases PC-1 and PC-2 were unaffected. Furthermore, TC-3-AGolf cells are resistant to serum starvation-induced apoptosis. Our findings suggest that Golf is involved in insulin status, cell survival, and regeneration of the insulin-secreting -cells during development and diabetes.