Partial depletion of serine/threonine protein kinase 25 (STK25), a member of the Ste20 superfamily of kinases, increases lipid oxidation and glucose uptake in rodent myoblasts. Here we show that transgenic mice overexpressing STK25, when challenged with a high‐fat diet, develop reduced glucose tolerance and insulin sensitivity compared to wild‐type siblings, as evidenced by impairment in glucose and insulin tolerance tests as well as in euglycemic‐hyperin‐sulinemic clamp studies. The fasting plasma insulin concentration was elevated in Stk25 transgenic mice compared to wild‐type littermates (4.9±0.8 vs. 2.6±0.4 ng/ml after 17 wk on high‐fat diet, P <0.05). Overexpression of STK25 decreased energy expenditure during the dark phase of observation ( P <0.05), despite increased spontaneous activity. The oxidative capacity of skeletal muscle of transgenic carriers was reduced, as evidenced by altered expression of Cpt1, Acox1 , and ACC. Hepatic triglycerides and glycogen were elevated (1.6‐ and 1.4‐fold, respectively; P <0.05) and expression of key enzymes regulating lipogenesis ( Fasn ), glycogen synthesis ( Gck ), and gluconeogenesis ( G6pc, Fbp1 ) was increased in the liver of the transgenic mice. Our findings suggest that overexpression of STK25 in conditions of excess dietary fuels associates with a shift in the metabolic balance in peripheral tissues from lipid oxidation to storage, leading to a systemic insulin resistance.—Cansby, E., Amrutkar, M., Mannerås Holm, L., Nerstedt, A., Reyahi, A., Stenfeldt, E., Borén, J., Carlsson, P., Smith, U., Zierath, J.R., Mahlapuu, M. Increased expression of STK25 leads to impaired glucose utilization and insulin sensitivity in mice challenged with a high‐fat diet. FASEB J. 27, 3660–3671 (2013). www.fasebj.org