Lactoferrin (LF) is a monomer glycoprotein in the mammalian colostrum that has multiple biological activities and a high affinity for iron ions. Not only does it have strong antibacterial activity, it also can regulate the release of cytokines in inflammatory areas, activate immune cells, and inhibit inflammatory diseases caused by nonpathogenic bacteria and the development of tumors. However, the anti-inflammatory mechanism of LF in inflammatory skin diseases induced by Propionibacterium acnes (P. acnes) has not been elucidated in vitro and in vivo. In this study, we investigated the effects of LF on the generation of inflammatory cytokines in HaCaT cells induced by heat-killed P. acnes. The expression of pro-inflammatory cytokines IL-8 increased after induction of HaCaT by heat-killed P. acnes, but it decreased significantly after LF treatment. Western blotting was used to examine the levels of TLR2, nuclear factor (NF) κB and intercellular cell adhesion molecule 1 protein induced by P. acnes in HaCaT cells, and the results showed that the levels were inhibited by LF. In addition, activated P. acnes (1 × 107 CFU/mL) was injected into the ears of experimental mice, which induced inflammation 24 hours after the injection. However, immunohistochemical analysis showed a significant reduction in the inflammatory response after LF treatment in the right ear relative to the untreated left ear, and the same result was seen with western blotting. In summary, this study revealed the treatment effect of LF on P. acnes-induced inflammation, thus providing support for the anti-acne properties of LF.