Lactate is now recognized as a regulator of fuel selection in mammals because it inhibits lipolysis by binding to the hydroxycarboxylic acid receptor 1 (HCAR1). The goals of this study were to quantify the effects of exogenous lactate on: 1) lipolytic rate or rate of appearance of glycerol in the circulation ( R a glycerol) and hepatic glucose production ( R a glucose), and 2) key tissue proteins involved in lactate signaling, glucose transport, glycolysis, gluconeogenesis, lipolysis, and β-oxidation in rainbow trout. Measurements of fuel mobilization kinetics show that lactate does not affect lipolysis as it does in mammals ( R a glycerol remains at 7.3 ± 0.5 µmol·kg −1 ·min −1 ), but strongly reduces hepatic glucose production (16.4 ± 2.0 to 8.9 ± 1.2 µmol·kg −1 ·min −1 ). This reduction is likely induced by decreasing gluconeogenic flux through the inhibition of cytosolic phosphoenolpyruvate carboxykinase (Pck1, alternatively called Pepck1; 60% and 24% declines in gene expression and protein level, respectively). It is also caused by lactate substituting for glucose as a fuel in all tissues except white muscle that increases glut4a expression and has limited capacity for monocarboxylate transporter (Mct)-mediated lactate import. We conclude that lipolysis is not affected by hyperlactatemia because trout show no activation of autocrine Hcar1 signaling (gene expression of the receptor is unchanged or even repressed in red muscle). Lactate regulates fuel mobilization via Pck1-mediated suppression of gluconeogenesis and by replacing glucose as a fuel. This study highlights important functional differences in the Hcar1 signaling system between fish and mammals for the regulation of fuel selection.