Abstract: To explore whether there is sexual dimorphism in hepatic glycogen metabolism in zebrafish (Danio rerio), we investigated it by morphological observation (transmission electron microscope), histological analysis, and liver glycogen (LG) content determination in liver of wild-type (WT), and found that the LG content in male zebrafish was significantly higher than the female (P<0.01). Subsequently, we performed histological analysis and LG determination in WT and stat5b mutant (stat5b^–/–) zebrafish. The results showed that the LG content in male stat5b^–/– was significantly lower than the control (P<0.05), whereas it was no significant difference in the female stat5b^–/– (P>0.05). It indicated that the function deficiency of stat5b reduced the sexual dimorphism in LG content. By RNA-seq analysis, we screened the differentially expressed genes (DEGs) and performed KEGG enrichment analysis with it. The results showed that these DEGs were significantly enriched in glucose metabolism-related pathways, in which gys2 (glycogen synthase 2) as a candidate gene potentially interacted with Stat5b. The experiment of transcription factor binding site (TFBS) prediction and dual-luciferase reporting system indicated that it had transcriptional activity in the predicted TFBS region, which demonstrated that Stat5b positively regulated gys2. Finally, the results showed that the LG level was significantly increased in stat5b overexpressed transgenic (stat5b-TG) male zebrafish (P<0.05), whereas there was no significant difference in female (P>0.05). Contrary to stat5b mutation, overexpression of stat5b increased the sexual dimorphism of LG content. These findings concluded that transcription factor Stat5b possibly regulated sexual dimorphism of LG synthesis through transcriptional control of gys2, thereby maintained liver glycogen metabolism in fish.