A 12-week experiment was conducted to evaluate the influences of thiamine on growth performance and intestinal mitochondrial biogenesis and function of M. amblycephala fed a high-carbohydrate (HC) diet. Fish (24.73 ± 0.45 g) were randomly assigned one of four diets; two carbohydrate levels (30 and 45%) and two thiamine levels (0 and 1.5 mg kg ⁻¹ ). HC diets significantly decreased DGC, GR MBW , FI MBW , intestinal activities of amylase, lipase, Na ⁺ , K ⁺ -ATPase, CK, complex I, III and IV, intestinal microvilli length, number of mitochondrial per field, ΔΨm, the P-AMPK/T-AMPK ratio, PGC-1β protein expression as well as the transcriptions of AMPKα1, AMPKα2, PGC-1β, TFAM, Opa-1, ND-1 and COX-1 and 2, while the opposite was true for ATP, AMP and ROS, and the transcriptions of Drp-1, Fis-1 and Mff. Dietary thiamine concentrations significantly increased DGC, GR MBW , intestinal activities of amylase, Na ⁺ , K ⁺ -ATPase, CK, complex I and IV, intestinal microvilli length, number of mitochondrial per field, ΔΨm, the P-AMPK/T-AMPK ratio, PGC-1β protein expression as well as the transcriptions of AMPKα1, AMPKα2, PGC-1β, Opa-1, ND-1, COX-1 and 2, SGLT-1 and GLUT-2. Furthermore, a significant interaction between dietary carbohydrate and thiamine was observed in DGC, GR MBW , intestinal activities of amylase, CK, complex I and IV, ΔΨm, the AMP/ATP ratio, the P-AMPK/T-AMPK ratio, PGC-1β protein expression as well as the transcriptions of AMPKα1, AMPKα2, PGC-1β, Opa-1, COX-1 and 2, SGLT-1 and GLUT-2. Overall, thiamine supplementation improved growth performance, and intestinal mitochondrial biogenesis and function of M. amblycephala fed HC diets.