A 60-day experiment was conducted to study the effect of dietary gelatinized (G) and non-gelatinized (NG) starch on the key metabolic enzymes of glycolysis (hexokinase, glucokinase, pyruvate kinase, and lactate dehydrogenase), gluconeogenesis (glucose-6 phosphatase and fructose-1,6 bisphosphatase), protein metabolism (aspartate amino transferase and alanine amino transferase), and TCA cycle (malate dehydrogenase) in Labeo rohita juveniles. In the analysis, 234 juveniles (2.53 +/- 0.04 g) were randomly distributed into six treatment groups each with three replicates. Six semi-purified diets containing NG and G cornstarch, each at six levels of inclusion (0, 20, 40, 60, 80, and 100) were prepared viz., T1 (100% NG, 0% G starch), T2 (80% NG, 20% G starch), T3 (60% NG, 40% G starch), T4 (40% NG, 60% G starch), T5 (20% NG, 80% G starch), and T6 (0% NG, 100% G starch). Dietary G:NG starch ratio had a significant (P < 0.05) effect on the glycolytic enzymes, the highest activities were observed in the T6 group and lowest in the T1 group. On the contrary, the gluconeogenic enzymes, the glucose-6-phosphatase and fructose-1,6 bisphosphatase activities in the organs, liver and kidney were recorded highest in the T1 group and lowest in the T6 group. The liver aspartate amino transferase activity showed an increasing trend with the decrease in the dietary G level. However, the muscle aspartate amino transferase activity was not significantly (P > 0.05) influenced by the type of dietary starch. The alanine amino transferase activity in both liver and muscle showed an increasing trend with the decrease in the dietary G level. The liver and muscle malate dehydrogenase activities were lowest in the T6 group and highest in the T1 group. Results suggest that NG (100%) starch diet significantly induced more the enzyme activities of amino acid metabolism, gluconeogenesis, and TCA cycle, whereas partial or total replacement of raw starch by gelatinized starch increased the glycolytic enzyme activity.