Objective: To explore the anti-fatigue effect of hemp seed oligopeptides, and its effect and mechanism between male and female individuals. Methods: Equal number of male and female mice were randomly divided into a blank control group, a positive control group and low, medium and high dose groups. The blank control group was given normal saline, the positive control group was given whey protein. Gavage treatment was carried out with the corresponding drug for 28 days. Body weight, exhaustive swimming time, blood lactic acid （BLA）, urea nitrogen （BUN）, liver glycogen （LG） contents of mice were measured. Results: Regardless of gender, flax seed oligopeptide had no significant effect on the body weight of mice, but could significantly prolong the exhaustive swimming time of mice, reduce the levels of blood lactic acid and blood urea nitrogen, and increase the content of liver glycogen. Considering gender factors, the body weight of female mice was significantly increased by medium dose of hemp seed oligopeptide, while the body weight of male mice was significantly decreased. Low dose of flax seed oligopeptide significantly prolonged exhaustive swimming time and decreased blood urea nitrogen content of male mice, but significantly increased body weight, exhaustive swimming time and liver glycogen content and decreased blood urea nitrogen content of female mice. High dose of hemp seed oligopeptide significantly increased the exhaustive swimming time and liver glycogen content, and significantly decreased the body weight and blood urea nitrogen content of male mice, while significantly increased the body weight, exhaustive swimming time and liver glycogen content, and significantly decreased the blood lactic acid value and blood urea nitrogen content of female mice. Conclusion: Hemp seed oligopeptide has anti-fatigue effect, and the dose has a significant effect on anti-fatigue effect in different genders.