The early diagnosis of colorectal cancer (CRC) is important because it is one of the most readily curable of all cancers, if detected early. However, the sensitivity of current markers is low. Immunostaining intensity for the monoclonal antibody Hb3 in CRC cell lines and tissues was stronger than in controls. Interestingly, this was associated with a low level of tumor differentiation. We used Hb3-coupled affinity chromatography to search for a corresponding Hb3 antigen as a candidate biomarker for early detection, and identified a Rho GTPase activating protein 6 (RhoGAP6) isoform 1 variant as an Hb3 antigen by mass spectrometry. Using reverse transcription polymerase chain reaction and western blot analysis, we confirmed that the expression levels of this variant were elevated in aberrant cells and tissues. Thus, the RhoGAP6 isoform 1 variant might serve as a biomarker for the development and progression of CRC.