Objective To investigate the relationship between mismatch repair (MMR) protein expression and microsatellite instability (MSI) and tumor TNM staging in colorectal cancer. Methods The clinical data of 1351 patients who underwent radical resection of colorectal cancer at the Xijing Digestive Hospital of Air Force Military Medical University from January 2008 to December 2017 were retrospectively analyzed. The MMR and MSI status in patients with different gender, age and TNM staging were analyzed. Results Of the 1351 specimens, 291 (22％) didn't express MMR protein. Univariate analysis showed that there were significant differences in the deletion rates of MSH2, MSH6 and PMS2 between patients ≥60 years old and<60 years old (all P<0.05);there were significant differences in the deletion rates of MSH2 between stage T3 and the other stages (P<0.05); the deletion rates of MLH1, PMS2 among different N stages, and the deletion rates of MSH2 and MSH6 between stage N0 or N1 and the other N stages were significantly different (all P< 0.05); the deletion rates of PMS2 among different M stages were significantly different (P< 0.05). There were significant differences in PMS2 deletion rates among different TNM stages and MLH1, MSH2 and MSH6 deletion rates between stage Ⅱ or Ⅲ and the other stages (all P< 0.05). There was significant difference in MSI positive rates between patients ≥60 years old and<60 years old (P<0.05); there were significant differences in MSI positive rates between stage T3 or T4 and the other T stages, among different N or M stages, and between TNM stage Ⅱ, ⅢorⅣand the other TNM stages (all P<0.05). Conclusions The MMR protein expression and MSI in colorectal cancer patients are closely related to tumor TNM staging. Detection of MMR protein expression and MSI in colorectal cancer patients has certain reference value for judging TNM staging of colorectal cancer. To a certain extent, it can guide the diagnosis and treatment of patients with colorectal cancer and judge the prognosis.