The aim of the present study was to investigate the effects of hemp seed oil supplementation on serum antioxidant indicators in dairy water buffalo. Eighteen dairy water buffaloes were randomly assigned to 3 treatments: (1) 0 g/d hemp seed oil supplement, (2)100 g/d hemp seed oil supplement, and (3) 200 g/d hemp seed oil supplement. The whole feeding experiment duration was 6 weeks with 2 weeks for adaptation and measurements were made at the end of whole feeding experiment. The results showed that, compared with group 1, hemp seed oil supplementation tended to increase the activity of total antioxidative capacity (T-AOC), glutathione peroxidase (GSH-PX) and catalase (CAT), and group 3 reached significance level (P < 0.05). Group 2 resulted in significantly decreased activity of peroxidase (MDA) compared with group 1, while group 3 had no significant effect. On the other hand, no significant differences were found between group 1 and the other treatments in the activity of peroxidase (POD) and superoxide dismutase (SOD). In conclusion, these results of this study indicated that supplement hemp seed oil would increase the antioxidant capacity of serum and enhance the immune function in organism of dairy water buffalo.