Obesity is an energy balance disorder in which intake is greater than expenditure, with most excess calories stored as triglyceride (TG). We previously reported that mice lacking the β‐isoform of protein kinase C (PKCβ), a diacylglycerol‐ and phospholipid‐dependent kinase, exhibit marked reduction in the whole body TG content, including white adipose tissue (WAT) mass. To investigate the role of this signaling kinase in metabolic adaptations to severe dietary stress, we studied the impact of a high‐fat diet (HFD) on PKCβ expression and the effect of PKCβ deficiency on profound weight gain. We report herein that HFD selectively increased PKCβ expression in obesity‐prone C57BL/6J mice, specifically in WAT; the expression levels were little or unchanged in the liver, muscle, kidney, and heart. Basal PKCβ expression was also found to be elevated in WAT of obese ob/ob mice. Remarkably, mice lacking PKCβ were resistant to HFD‐induced obesity, showing significantly reduced WAT and slightly higher core body temperatures. Unlike lean lipodystrophic mouse models, these mice did not have fatty livers, nor did they exhibit insulin resistance. Moreover, PKCβ^−/− mice exhibited changes in lipid metabolism gene expression, and such alterations were accompanied by significant changes in serum adipokines. These observations suggest that PKCβdeficiency induced a unique metabolic state congruous with obesity resistance, thus raising the possibility that dysregulation of PKCβ expression could contribute to dietary fat–induced obesity and related disorders. (HEPATOLOGY 2009.)