The present study was conducted to investigate the effects of dietary carbohydrate sources on insulin mediated glycemic responses and glucose homeostasis in black seabream. Four isonitrogenous (48%) and isolipidic (12%) diets were formulated to contain 20% glucose (GLU), sucrose (SUC), dextrin (DEX) and wheat starch (WS), respectively. The black seabream was randomly distributed into 12 tanks (n = 3) at a stocking density of 12 fish per tank. After 2 weeks of feeding, fishes were starved for 48 h then re-fed and sampled at 0, 1, 3, 6, 12, and 24 h. The results showed that the concentration of serum glucose increased at 1 h, peaked at 3 h, decreased at 12 h, and returned to basal level at 24 h in all dietary groups (p < 0.05). In contrast, there were significant differences in postprandial insulin levels among all treatments, especially in the DEX group, which showed higher insulin level at 3 h after feeding (p < 0.05). Compared with 0 h, liver glycogen in the WS and DEX groups increase significantly at 6 h and was depleted at 24 h (p < 0.05). Hepatic enzyme activities such as glucokinase (GCK) and pyruvate kinase (PK) were influenced by sampling time and dietary carbohydrate sources (p < 0.05). Fish fed with the DEX diet showed the highest hepatic GCK and PK activities and the lowest phosphoenol pyruvate carboxykinase (PEPCK) activity at 3 h among all the treatments. Slightly but not significantly higher phosphofructo kinase (PFK) activity was observed in fish fed with the SUC diet than those fed with the GLU diet at 6 h post prandial (p > 0.05). The relative expression of insulin signaling pathways such as insulin receptor substrate1 (irs1) and protein kinase B (akt1) were significantly up-regulated at 3 h in the DEX group (p < 0.05). Meanwhile, the mRNA expression levels of glucokinase (gk) and pyruvate kinase (pk) were sensitive to hepatic glycolysis, and their expressions were up-regulated at 1, 3, 6 h, especially at 3 h in the DEX group. Genes such as glucose 6 phosphatase (g6pc) and phosphoenol pyruvate carboxykinase (pepck) played an important role in hepatic gluconeogenesis, and their expressions were inhibited at 1, 3 h. Overall, the results of present study indicated that dietary carbohydrate sources resulted in a hyperglycaemic situation 3 h after feeding, most likely as a response to glucose loading. Moreover, glucose metabolic responses were regulated by different carbohydrate sources at different time points, and plays a important role in clearing glucose through increased activity of insulin, glycolysis, and glycogenesis, along with gluconeogenesis suppression.