Intermuscular bones (IBs) exist in the intermuscular septums of some species of teleosts. MsxC (muscle segment homeobox C) gene, a member of Hox gene family, was postulated to potentially play a role during the development of intermuscular bone in Hemibarbus labeo. In this study, we deleted MsxC gene in zebrafish genome by CRISPR/Cas9 technology to create the MsxC^-/- strain. Compared with wild-type zebrafish, MsxC^-/- zebrafish had shorter IBs in rear part of body, where both epineurals and epipleurals were shortened by more than 15% on average. However, there was no significant difference in the length of IBs in the anterior part of trunk. We also found less fast muscle fibers nearby septum in MsxC^-/- zebrafish, especially in rear part of body. On the contrary, more slow muscle fibers were found in MsxC^-/- zebrafish. Compared with wild type, the area of slow muscle in rear part of MsxC^-/- zebrafish was increased by 2.22%(P<0.01), whereas the area of trunk anterior part was increased by 0.97% (P<0.05). All these results suggested that the deletion of MsxC gene affected both the formation of IBs and the development of axial muscle.