This study aimed to investigate the effects of dietary betaine supplementation on lipid metabolism, immunity, and testicular oxidative status in Hu sheep. Experimental sheep were fed with betaine-containing diets, a basal diet supplemented with 0 g/d (control group), 1 g/d (B1), and 3 g/d betaine (B2). The serum concentration of triglycerides was increased in Hu sheep receiving the diet supplemented with betaine. Regarding granulocytes, the ratio of monocytes, neutrophils and basophils was increased (P < 0.05). ELISA results illustrated that testicular CAT (P < 0.05), SOD (P < 0.01), and GSH-Px activity (P < 0.05) was higher, while MDA content was significantly decreased (P < 0.01) after fed with betaine-supplemented diets. qPCR results demonstrated that the mRNA expression of CAT (P < 0.05), SOD2 (P < 0.01) and GSH-Px (P < 0.05) was upregulated in B1 group, significantly upregulated (P < 0.01) in B2 group relative to the control group, which showed the same trend with its protein expression as reflected by Western blot. Furthermore, PCNA expression was lower (P < 0.05) in the testis of Hu sheep than control group. Moreover, LKB1 expression was increased (P < 0.01), and mRNA expression of AMPK was decreased (P < 0.05) in B1 group. The relative gene expression of mTOR was significantly higher (P < 0.01) in B2 group than control group. RAPTOR expression was increased in B1 (P < 0.05) and B2 (P > 0.05) group. Western blot revealed that the ratio of P-mTOR and mTOR was significantly increased (P < 0.01) after fed with betaine-supplemented diets. In conclusion, betaine supplementation improved serum lipid metabolism, immune response, and increased the testicular antioxidant capacity of Hu sheep, which might be regulated via mTOR signaling pathway.