The present study was aimed at evaluating the expression of heat shock protein 27 (HSP27) and heterogeneous nuclear ribonucleoprotein K (hnRNP K), two potential biomarkers of many cancers, in colorectal cancer (CRC) and their clinical significance. Expression of HSP27 and hnRNP K were investigated by immunohistochemistry (IHC) in a series of tissue microarrays containing 175 primary colorectal cancers and their corresponding normal mucosa samples and matched with clinicopathological features and patient survival. HSP27 and hnRNP K displayed more frequent strong immunoreactivity in primary colorectal tumor samples compared with adjacent non-cancer tissue (P < 0.001). Increased cytoplasmic expression of HSP27 and hnRNP K were associated with tumor location (P = 0.032 and P < 0.001, respectively), poorer overall survival (P = 0.004 and P = 0.02, respectively) and to an unfavorable prognosis for CRC patients in multivariate analysis (P = 0.019 and P = 0.01, respectively). Their overexpression combination identified a subset of patients with definitively worse prognosis than any other combination (P < 0.001). Overexpression of HSP27 and hnRNPK were independent markers of poor prognosis, and their combination predicted definitively adverse outcomes in CRC patients.