Objective: To investigate the expression and clinical significance of osteopontin (OPN) and matrix metalloproteinase-3 (MMP-3) in rectal adenocarcinoma. Methods: Immunohistochemical EnVision method was used to detect the expression and correlation of OPN and MMP-3 in 64 cases of rectal adenocarcinoma and adjacent tissues. The Clinical pathological features were compared and follow-up. Results: The expressions of OPN and MMP-3 in rectal adenocarcinoma were significantly higher than those in adjacent tissues, and the expressions of OPN and MMP-3 were correlated with clinical stage, differentiation and lymph node metastasis (P<0.05). There was no correlation with age, gender, and depth of invasion (P>0.05), and there was no significant difference. Spearman rank correlation analysis showed that OPN was positively correlated with MMP-3 expression in rectal adenocarcinoma (r=0.461, P<0.05). The clinical stage, differentiation degree and lymph node metastasis of rectal adenocarcinoma were correlated with the positive expression of OPN and MMP-3 (P<0.05), and there were significant differences. The overall survival rate of OPN and MMP-3 positive patients was lower than that of negative patients (P<0.05). There was a significant difference. Conclusion: The expressions of OPN and MMP-3 in rectal adenocarcinoma are significantly increased, and the expression is positively correlated. It is closely related to clinical stage, differentiation, lymph node metastasis and prognosis, and may be an important biomarker for rectal adenocarcinoma. OPN and MMP-3 are important for the diagnosis and treatment of rectal adenocarcinoma.