Fructose is a unique nutrient, due in part to its selective metabolism in the liver. Diets enriched in fructose or sucrose induce a hepatic stress response characterized by activation of c-jun N-terminal kinase. The aim of this study was to examine the regulation of c-jun N-terminal kinase by fructose in rat primary hepatocytes. Fructose was provided to rat primary hepatocytes using a fructose regenerating system, consisting of inulin and inulinase. This system provides a more physiologic delivery of fructose and avoids large disturbances in hepatocyte ATP concentrations. Fructose delivery increased c-jun N-terminal kinase activity and serine 307 phosphorylation of insulin receptor substrate-1 and reduced tyrosine phosphorylation of insulin receptor substrate-1. Activation of c-jun N-terminal kinase was maximal at a fructose concentration of 0.6 mmol/L. Fructose delivery did not increase the phosphorylation of p38 mitogen-activated protein kinase, extracellular signal regulated kinase, c-jun, or activating transcription factor-2, the latter 2 downstream nuclear targets of c-jun N-terminal kinase. However, fructose delivery increased the phosphorylation of mitogen-activated protein kinase kinase-7 (MKK7), an upstream activator of c-jun N-terminal kinase, and the association of c-jun N-terminal kinase with c-jun N-terminal kinase-interacting protein-1, a scaffold protein that can sequester protein signaling complexes in the cytosol. These data suggest that fructose may selectively activate c-jun N-terminal kinase via regulation of MKK7 and scaffold proteins.