This study was conducted to investigate the effects of magnesium (Mg) supplementation on the growth performance, muscle fiber development and flesh quality of Megalobrama amblycephala fed a high-carbohydrate (CHO) diet. In vivo, six experimental diets, a control diet (30% CHO, C), and five Mg supplementation levels (0 (HC1), 200 (HC2), 400 (HC3), 800 (HC4) and 1200 (HC5) mg kg−1, respectively) of high-CHO diet (45% CHO), were formulated and then fed to M. amblycephala (initial weight 43.15 ± 0.43 g) for 13 weeks. Results showed that the HC1 diets remarkably decreased weight gain rate (WGR), specific growth rate (SGR), water holding capacity (WHC), pH 24 h postmortem, collagen content, textural properties (hardness, tenderness, chewiness and resilience), muscle fiber density, succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) activities, as well as transcriptions of ampkα2, pgc-1α, mrf-4, myf-5 and myf-6 compared with the C group, but muscle AMP/ATP ratio, ATPase activities and mstna and mstnb transcription showed an opposite trend. The HC3 group of fish obtained relatively high values of WGR, SGR, muscle lipid and protein content, collagen content, textural properties (hardness, tenderness, chewiness and resilience), fiber density, SDH and MDH activities, AMP/ATP ratio as well as transcriptions of ampkα1, pgc-1α, myod, myog and myf-5 compared with other HC groups. In vitro, the isolated primary hepatocytes from M. amblycephala were assigned into four experimental groups: a control group (5.5 mM glucose, Control), a high glucose group (30 mM glucose, HG), an AICAR group (30 mM glucose + 250 μM AICAR, HGA) and an MgSO4 group (30 mM glucose + 2 mM MgSO4, HGM). Results showed that MgSO4 treatment remarkably increased p-Ampk/t-Ampk ratio, Pgc-1α protein content and transcriptions of ampkα1, ampkα2 and pgc-1α compared with the HG group. Overall, based on regression analysis for SGR, the optimal Mg level of M. amblycephala fed high-CHO diets was 366.67 mg kg-1, which was consistent with the above results that fish fed HC3 diets displayed higher the growth performance, muscle fiber density and flesh quality of M. amblycephala.