Prostatitis, defined as a pathological inflammatory change in the prostate tissue, is one of the most prevalent urological conditions in men. However, optimal management of prostatitis remains unclear, and treatment outcomes are unsatisfactory owing to adverse effects. Carica papaya leaf extract (PAL) is known for its antioxidant, immunomodulatory, and anticancer properties; however, evidence of its anti-inflammatory effect in prostatic tissues remains elusive. In this study, the therapeutic effects and underlying molecular mechanisms of PAL in mice with experimental autoimmune prostatitis (EAP) and a prostatic cell line (RWPE-1 cells) exposed to inflammatory conditioned medium were investigated. PAL suppressed pathological alterations in EAP and markedly reduced prostate weight in EAP mice. Histological analysis revealed that PAL alleviates prostatic hyperplasia. Furthermore, PAL significantly reduced cyclooxygenase-2 mRNA and protein expression; production of inflammatory cytokines, including interleukin-6, tumor necrosis factor-α, and transforming growth factor-β; and TRAF6/TAK1/MEK/ERK and NF-κB pathway-related protein expression. TRAF6/TAK1/MEK/ERK and NF-κB pathway-related proteins were upregulated in inflammatory conditioned medium-stimulated RWPE-1 cells, but PAL reduced the expression of these markers. Particularly, PAL treatment suppressed the nuclear translocation of NF-κB p65 and phosphorylation of p65 in RWPE-1 cells exposed to the inflammatory conditioned medium. Collectively, the results demonstrate the anti-proliferative and anti-inflammatory effects of PAL in the experimental prostatitis model, which highlights the potential of PAL as a new therapeutic agent in the treatment of prostatic disease.