OBJECTIVE—Guanine nucleotide binding protein (G protein)–mediated signaling plays major roles in endocrine/metabolic function. Regulators of G protein signaling (RGSs, or RGS proteins) are responsible for the subsecond turn off of G protein signaling and are inhibitors of signal transduction in vitro, but the physiological function of RGS proteins remains poorly defined in part because of functional redundancy.RESEARCH DESIGN AND METHODS—We explore the role of RGS proteins and Gαi2 in the physiologic regulation of body weight and glucose homeostasis by studying genomic “knock-in” mice expressing RGS-insensitive Gαi2 with a G184S mutation that blocks RGS protein binding and GTPase acceleration.RESULTS—Homozygous Gαi2G184S knock-in mice show slightly reduced adiposity. On a high-fat diet, male Gαi2G184S mice are resistant to weight gain, have decreased body fat, and are protected from insulin resistance. This appears to be a result of increased energy expenditure. Both male and female Gαi2G184S mice on a high-fat diet also exhibit enhanced insulin sensitivity and increased glucose tolerance despite females having similar weight gain and adiposity compared with wild-type female mice.CONCLUSIONS—RGS proteins and Gαi2 signaling play important roles in the control of insulin sensitivity and glucose metabolism. Identification of the specific RGS proteins involved might permit their consideration as potential therapeutic targets for obesity-related insulin resistance and type 2 diabetes.