Objective:To establish an animal model of tooth separation, and analyze the expression of substance P in dental pulp. Methods:0.03 mm wire were applied between the right maxillary first molar and second molar of the rats. The dental pulp were stained by HE and immunochemistry to observe the expression of substance P(SP) after 6 h,12 h,1 d,3 d and 7 d, respectively. Results:Expression of SP was increased after 6 h,12 h and reached the peak after 1 d,and then decreased. The expression of SP was almost the same as normal after 7 d. Conclusion:The change of SP in dental pulp during orthodontic tooth separation indicated that SP may play an important role in orthodontical pain.