[Purpose] To investigate the correlation between the effect treadmill exercise and change in serum proteins in rats with osteoarthritis, a study of proteins was carried out using a mass spectrometer. [Subjects and Methods] Rats were randomly divided into five groups. After 4 weeks of treadmill training, serum from each rat was analyzed by Liquid chromatography-electrospray ionization tandem mass spectrometry. Complementary component 9 (C9) was discovered to be downregulated in the serum of the exercise groups, and this was validated by Western blot. [Results] Seventeen proteins were discovered to be elevated in the monosodium iodoacetate injection osteoarthritis group samples by more than 1.5 fold compared with the control group. One of the proteins upregulated, C9 protein, was validated, and it was found to decrease in the middle-intensity exercise group. [Conclusion] We showed that the serum level of C9, an inflammatory-related protein, decreased after treadmill exercise. Therefore, treadmill exercise with an appropriate intensity might be recommended for OA patients.