VSTM1 (V-set and transmembrane domain containing 1) is a novel membrane molecule identified from immunogenomics, which has two main isoforms, VSTM1-v1 and VSTM1-v2. VSTM1-v1 is a type I transmembrane protein, and VSTM1-v2 is a classical secretory protein, lacking only the transmembrane domain compared with VSTM1-v1. This study was designed to generate VSTM1-specific monoclonal antibodies (MAbs) for further exploration of its expression and function. Mice were immunized with two recombinant prokaryotic proteins of GST-VSTM1-v2 and VSTM1-v2 without any tag. Hybridomas were generated by the fusion of the splenocytes to Sp2/0 myeloma cells. Three hybridoma cell lines (2C11, 6E11, and 7A8) stable in secreting anti-VSTM1 MAb were obtained and further characterized. All three MAbs were IgG2b isotype and effective in detecting the overexpressed VSTM1 in both Western blot and flow cytometry assays, while recognizing the endogenous VSTM1 in Western blot analysis only. These MAbs could be helpful in the basic study of VSTM1 and in revealing the interesting conformation difference between the overexpressed and endogenous proteins.