In rainbow trout, dietary carbohydrates are poorly metabolized compared to other macronutrients. One prevalent hypothesis suggests that high dietary amino acid levels could contribute to the poor utilization of carbohydrates in trout. In mammals, alanine is considered an important gluconeogenic precursor, but has recently been found to stimulate AMP-activated protein kinase (AMPK) to reduce glucose levels. In trout, the effect of alanine on glucose fluxes is unknown. The goal of this study was to determine the effects of 4h exogenous alanine infusion on glucose metabolism in rainbow trout. Glucose fluxes, glucose appearance (Ra), and disposal (Rd) were measured in vivo. Key glycolytic and gluconeogenic enzyme expression and activity and cell signaling molecules relevant to glucose metabolism were assessed in liver and muscle. Results show that alanine inhibits Ra glucose (from 13.2+/-2.5 to 7.3+/-1.6 micromol / kg min) and Rd glucose (from 13.2+/-2.5 to 7.4+/-1.5 micromol / kg min) and the slight mismatch between Ra and Rd caused a reduction in glycemia, similar to the effects of insulin in trout. The reduction in Rd glucose can be partially explained by a reduction in glut4b expression in red muscle. In contrast to mammals, alanine-dependent glucose-lowering effects in trout did not involve hepatic AMPK activation, suggesting a different mechanistic basis. Interestingly, protein kinase B (AKT) activation increased only in muscle similar to effects observed in insulin-infused trout. We speculate that alanine-dependent effects were probably mediated through stimulation of insulin secretion which could indirectly promote alanine oxidation to provide the needed energy.