Purpose: To investigate the intestinal absorption characteristics of berberine hydrochloride (BBH) under different perfusion conditions in rats.Methods: Based on the in situ single-pass intestinal perfusion model of rats, HPLC was used to determine the content of berberine hydrochloride in solution after perfusion under different conditions. The absorption rate constant (Ka), effective permeability coefficient (Papp) and cumulative absorption per unit area (Q) under different perfusion conditions were analyzed by one-way ANOVA.Results: The Papp and Ka of BBH in perfusion solution at pH 7.4 were greater than those in perfusion solution at pH 6 and 8. There was no significant difference (p > 0.05) in Papp and Ka of duodenum, jejunum and ileum at high, medium and low concentrations of berberine hydrochloride perfusion solution. The Q increased linearly with increase of mass concentration of perfusion solution. The Ka and Papp of BBH in duodenum, jejunum, and ileum of BBH and berbamine hydrochloride (BAH) combined at different ratios were higher than those of BBH control group at the same BBH concentration, but absorption of BBH in the ratio B40:A50 and B30:A20 groups was highest. In the ratio of B40:A50 ratio, B30:A20 ratio group or the same concentration's BBH group, Ka and Papp of BBH decreased in the order of jejunum > duodenum > ileum.Conclusion: Berberine hydrochloride is absorbed in neutral environment of pH 7.4. The intestinal absorption mechanism of BBH is passive diffusion, and jejunum is the best intestinal segment for absorption. BAH promotes the absorption of BBH.