Colorectal cancer (CRC) is the third most common cancer worldwide and has poor prognosis. To identify the proteins involved in colorectal carcinogenesis, we employed 2-DE and MALDI-TOF/TOF-based proteomics approach to study the differentially expressed proteins in tumor and adjacent nontumor tissue samples. Samples from 10 colorectal patients were analyzed. Of the 7 significantly and consistently altered proteins identified, hnRNP A1 was one of the most significantly altered proteins and its overexpression was confirmed using RT-PCR and Western blot analyses. Immunohistochemical examination showed that the enhanced expression of hnRNP A1 was correlated with the increasing severity of colorectal tissue and the progression of the colorectal cancer, as well as UICC (International Union against Cancer) staging, histo-differentiation, recurrence and decreased survival. By developing a highly sensitive immunoassay, hnRNP A1 could be detected in human serum and was significantly elevated in CRC patients compared with healthy volunteers. We proposed that hnRNP A1 could be considered as a novel serum tumor marker for CRC that may have significance in the detection and in the management of patients with this disease. Knockdown of hnRNP A1 expression by RNA interference led to the significant suppression of the cell growth in colorectal cancer SW480 cells in vitro. These data suggested that hnRNP A1 may be a potential biomarker for early diagnosis, prognosis, and monitoring in the therapy of colorectal cancer. Further studies are needed to fully assess the potential clinical value of this biomarker candidate.