© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.The black pigmentation-related genes in Porphyromonas gingivalis are primarily involved in regulating gingipain functions. In this study, we identified a pigmentation-related gene, designated as pgn_0361. To characterize the role of pgn_0361 in regulating P. gingivalis-mediated epithelial cell detachment and inhibition of wound closure, PgΔ0361, an isogenic pgn_0361-defective mutant strain, and PgΔ0361C, a complementation strain, were constructed using P. gingivalis ATCC 33277. The gingipain and hemagglutination activities, as well as biofilm formation, were examined in all three strains. The effect of P. gingivalis strains on epithelial cell detachment was investigated using the HO-1-N-1 and Ca9-22 epithelial cell lines. The inhibition of wound closure by heat-killed P. gingivalis cells and culture supernatant was analyzed using an in vitro wound closure assay. Compared to the wild-type strain, the PgΔ0361 strain did not exhibit gingipain or hemagglutination activity but exhibited enhanced biofilm formation. Additionally, the PgΔ0361 strain exhibited attenuated ability to detach the epithelial cells and to inhibit wound closure in vitro. Contrastingly, the culture supernatant of PgΔ0361 exhibited high gingipain activity and strong inhibition of wound closure. The characteristics of PgΔ0361C and wild-type strains were comparable. In conclusion, the pgn_0361 gene is involved in regulating gingipains. The PGN_0361-defective strain exhibited reduced virulence in terms of epithelial cell detachment and inhibition of wound closure. The culture supernatant of the mutant strain highly inhibited wound closure, which may be due to high gingipain activity.