High-fructose consumption is associated with insulin resistance and diabetic dyslipidemia, but the underlying mechanism is unclear. We show in hamsters that high-fructose feeding stimulated forkhead box O1 (FoxO1) production and promoted its nuclear redistribution in liver, correlating with augmented apolipoprotein C-III (apoC-III) production and impaired triglyceride metabolism. High-fructose feeding upregulated peroxisome proliferator-activated receptor-γ coactivator-1β and sterol regulatory element binding protein-1c expression, accounting for increased fat infiltration in liver. High-fructose-fed hamsters developed hypertriglyceridemia, accompanied by hyperinsulinemia and glucose intolerance. These metabolic aberrations were reversible by fenofibrate, a commonly used anti-hypertriglyceridemia agent that is known to bind and activate peroxisome proliferator-activated receptor-α (PPARα). PPARα physically interacted with, but functionally antagonized, FoxO1 in hepatic apoC-III expression. These data underscore the importance of FoxO1 deregulation in the pathogenesis of hypertriglyceridemia in high-fructose-fed hamsters. Counterregulation of hepatic FoxO1 activity by PPARα constitutes an important mechanism by which fibrates act to curb apoC-III overproduction and ameliorate hypertriglyceridemia.