The cell surface transmembrane 4 superfamily member 5 protein (TM4SF5) has been implicated in various human cancers. Immunization with a peptide vaccine targeting human TM4SF5 has been shown to exert prophylactic and therapeutic effects against the development of hepatocellular carcinoma and colon cancer in mouse models. In this study, we developed a novel monoclonal antibody (mEC2‑CF) targeting a cyclic epitope of TM4SF5 and evaluated its reactivity to TM4SF5 in colorectal cancer (CRC) cells and cancer tissues. The isotype of mEC2‑CF was IgG2a and the antibody specifically recognized the cyclic peptide, based on ELISA. The antibody recognized recombinant TM4SF5 overexpressed in 293F cells, irrespective of N‑glycosidase F treatment. The antibody was internalized into the cytosol after binding to the surface of TM4SF5‑expressing CRC cells, suggesting that this antibody may be useful in therapeutics. In addition, we evaluated TM4SF5 expression in the tissues of patients with CRC patients to determine its prognostic significance. TM4SF5 expression was assessed by immunohistochemistry using mEC2‑CF and tissue microarray blocks of 204 primary CRC samples. The overall rate of TM4SF5 overexpression in the samples (immunohistochemical score >4) was 27.0% (55 of 204). The increased expression of TM4SF5 was significantly associated with a shorter survival rate (P=0.0014) and a worse disease‑free survival (P=0.0483) of patients with CRC. No association was observed between TM4SF5 expression and clinicopathological characteristics, apart from tumor depth of invasion (P=0.027). These results suggest that our novel antibody can be used to detect endogenous and recombinant TM4SF5, and that TM4SF5 may be a possible marker for the poor prognosis of patients with CRC.