Osteopontin (OPN) is a phosphorylated glycoprotein which is associated with tumor progression, development, and metastasis. Recently, it has been reported that OPN is highly upregulated in a variety of human malignancies. The aim of this study is to investigate the clinical significance of OPN mRNA expression in colorectal cancer (CRC). Conventional reverse transcription polymerase chain reaction (RT-PCR) and Western blot assays were performed to detect the expression of OPN mRNA and protein in human CRC cell lines and normal cell line. Real-time quantitative RT-PCR assay was performed to analyze the expression of OPN mRNA in 82 CRC tissue samples and corresponding non-tumor tissues. Immunohistochemistry was also performed to detect the expression of OPN protein in above tissues. Finally, the correlation between the status of OPN mRNA expression and clinicopathological factors and clinical outcome was evaluated. Compared with normal human intestinal epithelial cell line, human CRC cell lines showed high level of OPN gene expression at both transcriptional and translational levels. Moreover, the results of real-time quantitative RT-PCR and immunohistochemistry showed that the expression levels of OPN mRNA and protein in tumor tissues were significantly higher than those in the corresponding non-tumor tissues (P < 0.001). The expression level of OPN mRNA was significantly correlated with lymph node metastasis, lymphatic or venous invasion, and TNM stage (P = 0.0033, 0.0061, 0.0008, and 0.0012, respectively). Moreover, we also observed that the disease-free and overall survival rates in patients with high OPN mRNA expression were significantly shorter than those in patients with low OPN mRNA expression (P = 0.0047 and 0.0125). Additionally, the status of OPN mRNA expression was an independent prognostic factor for the prognosis of CRC patients (P = 0.008; RR, 2.775; 95% confidence interval, 2.334-3.811). OPN might play an important role in CRC progression and the status of OPN mRNA expression could be a novel prognostic molecular marker for CRC patients.