Propionibacterium acnes (P. acnes) has been implicated in the progression of acne inflammation. Because current acne medications have various side effects, it is necessary to explore alternative medications possessing anti-inflammatory activity against P. acnes. We investigated the inhibitory effects of polyphyllin I (PPI) on P. acnes-induced inflammation in vitro. In this study, we examined the effects of PPI on the production of inflammatory cytokines in HaCaT keratinocytes treated with heat-killed P. acnes. These treated HaCaT keratinocytes showed increased expression of Toll-like receptor 2 (TLR2) and production of inflammatory cytokines. PPI significantly suppressed the secretion of inflammatory cytokines, including interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-, and the expression of TLR2 in P. acnes-treated cells. Moreover, we studied the influence of PPI on the nuclear factor-B (NF-B) and mitogen-activated protein kinase (MAPK) signaling pathways in P. acnes-treated keratinocytes. PPI diminished the activation of NF-B. Phosphorylated p38 levels were markedly increased after treatment with heat-killed P. acnes but were decreased after treatment with PPI, while the effect of PPI on ERK phosphorylation was not significant. Heat-killed P. acnes and PPI did not have any effect on JNK phosphorylation. Furthermore, we confirmed that NF-B p65 inhibitor (BAY11-7082), p38 MAPK inhibitor (SB203580), and PPI blocked the expression of IL-8 in heat-killed P. acnes-treated cells. These results demonstrated that PPI has potential for development as a treatment for acne inflammation.