There is currently no licensed vaccine against Chlamydia trachomatis, the leading cause of sexually transmitted bacterial disease worldwide. Conventional vaccination attempts using surface-exposed chlamydial antigens have achieved only partial success. We have employed a novel vaccination strategy using a secreted protein, chlamydial protease-like activity factor (CPAF), which has been shown to degrade host major histocompatibility complex transcription factors and keratin-8 and therefore may allow immune evasion and establishment of a productive infection. Intranasal immunization using recombinant CPAF (rCPAF) plus interleukin-12 (IL-12) (rCPAF+IL-12 immunization) was used to assess the protective immunity against genital Chlamydia muridarum infection in BALB/c mice. rCPAF+IL-12 immunization induced robust gamma interferon (IFN-gamma) production and minimal IL-4 production by splenocytes upon in vitro recall with rCPAF. The total and immunoglobulin G2a (IgG2a) anti-rCPAF antibody levels in serum were significantly elevated after rCPAF+IL-12 vaccination, as were the total antibody, IgG2a, and IgA levels in bronchoalveolar lavage and vaginal fluids when the animals were compared to animals that received rCPAF alone. rCPAF+IL-12-vaccinated mice displayed significantly reduced bacterial shedding upon chlamydial challenge and accelerated resolution of infection compared to mock-immunized (phosphate-buffered saline) animals. Moreover, rCPAF+IL-12-immunized animals exhibited protection against pathological consequences of chlamydial infection, including the development of hydrosalpinx and oviduct dilatation. This vaccination regimen also reduced the development of fibrosis and the influx of neutrophils into the upper genital tract when the animals were compared to mock-immunized (phosphate-buffered saline) animals after bacterial challenge. rCPAF+IL-12-mediated resolution of the bacterial infection and protection against Chlamydia-induced inflammatory disease were highly dependent on endogenous IFN-gamma production. Together, these results demonstrate that secreted chlamydial antigens may be novel vaccine candidates to induce protective immunity.