ABSTRACTMycoplasma genitaliumis a prokaryotic microorganism that causes urogenital tract infections.M. genitaliumprotein of adhesion (MgPa) was essential forM. genitaliumattachment and subsequent invasion into host cells. Our prior research confirmed that Cyclophilin A (CypA) was the binding receptor for MgPa and MgPa-CypA interaction can lead to the production of inflammatory cytokines. In this study, we revealed that the recombinant MgPa (rMgPa) could inhibit the CaN-NFAT signaling pathway to reduce the level of IFN-γ, IL-2, CD25, and CD69 in Jurkat cells by binding to the CypA receptor. Moreover, rMgPa inhibited the expressions of IFN-γ, IL-2, CD25, and CD69 in primary mouse T cells. Likewise, the expressions of these T cells activation-related molecules in CypA-siRNA-transfected cells and CypA−/−mouse primary T cell was strengthened by rMgPa. These findings showed that rMgPa suppressed T cell activation by downregulating the CypA-CaN-NFAT pathway, and as a result, acted as an immunosuppressive agent.IMPORTANCEMycoplasma genitaliumis a sexually transmitted bacterium that can co-infect with other infections and causes nongonococcal urethritis in males, cervicitis, pelvic inflammatory disease, premature birth, and ectopic pregnancy in women. The adhesion protein ofM. genitalium(MgPa) is the primary virulence factor in the complicated pathogenicity ofM. genitalium. This research proved that MgPa could interact with host cell Cyclophilin A (CypA) and prevent T cell activation by inhibiting Calcineurin (CaN) phosphorylation and NFAT nuclear translocation, which clarified the immunosuppression mechanism ofM. genitaliumto host T cells. Therefore, this study can provide a new idea that CypA can be used for a therapeutic or prophylactic target forM. genitaliuminfection.