The present study aimed to investigate the expression of the Hedgehog (Hh) signaling pathway between microsatellite-unstable (MSI) and microsatellite-unstable (MSS) colorectal cancers (CRCs). A total of 61 samples of CRC tissue and corresponding blood samples were obtained from the surgical department of our hospital. The tissue samples were examined by immunohistochemistry using antibodies against Sonic Hh (SHH), Pathed (PTCH) and Gli1, and evaluated independently for protein expression by two pathologists blinded to clinical outcome. Based on the immunohistochemistry results, SHH and PTCH expression varied in terms of histological type. In mucinous adenocarcinoma (MA) Hedgehog signaling was not highly expressed. There were more significant differences in the expression of SHH and PTCH (P<0.05), compared with Gli1. Moreover, significant differences were found in the expression of SHH, Gli1 and PTCH between the MSI and MSS groups (P<0.05). Hedgehog signals were more frequently expressed in the MSI group compared with the MSS group. In conclusion, this study indicates that the expression of the Hh signaling pathway may play a significant role in MSI in colorectal cancer.