To observe the hemp seed pill on defecation function, pepsin activity and lymphocyte proliferation on rat constipation model. Methods: Seventy two rats were randomly divided into three groups: blank control group, model group and treatment group, twenty four rats in each group. The model group and the treatment group were used to establish constipation model. The blank control group and model group were not given intervention. The treatment group was given 10 hemp seed pills by gavage and in drinking water, once a day for a week. The intestine propulsion rate was calculated by ink propelling test to observe the defecation function of rats; Hematoxylin and eosin (H&E) stains were observed under the microscope to observe the conditions of intestinal tract; Pepsin activity was tested and lymphocyte proliferation was observed by using MTT and flow cytometry. Results: 1) Compared with the blank control group, fecal number, fecal weight, particle weight and the intestine propulsion rate in the model group and the treatment group decreased (P<0.05); Compared with the model group, the defecation function of the treatment group was significantly improved (P<0.05). 2) Compared with the blank control group, the inflammatory cell infiltration in colon and the thinning layer of muscle were observed in the model group. After drug treatment, the inflammation was improved and the muscle layer was thicken. 3) Compared with the blank control group, 2 h gastric juice volume and pepsin activity in the model group and the treatment group decreased significantly (P<0.05); Compared with the model group, 2 h gastric juice volume and pepsin activity in the treatment group increased significantly (P<0.05). 4) Compared with the blank control group, the lymphocyte proliferation (P<0.05), including the percentage of CD4+, CD8+ T-cells and T-lymphocyte subsets (CD4+/CD8+) ratios in model group and treatment group decreased (P<0.05); Compared with the model group, the lymphocyte proliferation (P<0.05), including the percentage of CD4+, CD8+ T-cells and T-lymphocyte subsets (CD4+/CD8+) ratios in the treatment group increased (P<0.05). Conclusion: Hemp seed pill can effectively improve defecation function and enhance the enzymatic activity of pepsin, which is relevant with the increasing lymphocyte proliferation, especially with the percentage of CD4+, CD8+ T-cells, and T-lymphocyte subsets (CD4+/CD8+) ratios.