Transglutaminase 4 (TG4) is a member of the enzyme family that catalyzes the calcium-dependent post-translational modification of proteins via cross-linking, polyamination, or deamidation. TG4 exhibits prostate-specific expression pattern and plays a crucial role in the formation of the copulatory plug in rodents. However, the physiological function(s) of human TG4 remains speculative. Human TG4 has been postulated to participate in the maturation process of sperm by modifying its cell surface, which results in suppression of sperm antigenicity in the female genital tract. To better understand the pathophysiological role of TG4 in prostate tissue, we generated monoclonal antibodies (MAb) against human TG4 in mice by repeated injections with the recombinant human TG4. Western blot analysis demonstrated that the selected MAbs react specifically with TG4, but not with other isoenzymes of the TG family. Immunocytochemical and immunohistochemical analyses showed that specific staining is observed with the cells overexpressing TG4 and with the paraffin-embedded prostate tissue specimens obtained from the benign prostate hyperplasia and prostate cancer patients, respectively. Our results indicate that these MAbs are suitable for detecting TG4 in the cultured cells or prostate tissues for investigating the biological functions of human TG4.