Objective To explore the roles of histamine and tryptase in chronic pancreatitis pain development and maintenance. Methods Rat models of chronic pancreatitis were prepared, and the histamine and tryptase contents were examined in the pancreatic specimens of both model group and the control group. The pancreas incubation supernatants of both groups were injected into the plantar surface of the rat hindpaw, and then the pain thresholds were observed at 0.5, 1, 3, 5, and 7 h after injection. Rats injected with the supernatant of chronic pancreatitis model were also given H1 receptor blocker or protease inhibitor to observe their treatment for hyperalgesia. Results The histamine and tryptase contents in the pancreas specimens were increased in chronic pancreatitis model rats. Injection of supernatant from the chronic pancreatitis specimens had a profound hyperalgesia effect within 5 h after injection, and the effect could be partially reduced by H1 receptor blockers and protease inhibitors. Conclusion Our findings suggest that histamine and tryptase are important mediators in the development and maintenance of the chronic pancreatitis pain.