Porcine placental extract (PPE) is used as a nonprescription drug for analeptics and in health foods and cosmetics in Japan, Korea and China. It was reported that PPE has anti-oxidative and anti-inflammatory activities; however, the mechanisms and the responsible molecules involved in these activities are still unclear. Here, we investigated how enzymatically prepared PPE affects proinflammatory factors such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in a cultured macrophage cell line, RAW264.7, when co-stimulated with lipopolysaccharide (LPS). Enhanced production of IL-1β, IL-6 and TNF-α by LPS was significantly reduced by the addition of PPE and these effects were dose dependent. Nitric oxide (NO) production induced in cultured macrophages by LPS was also inhibited by PPE. Real-time PCR after the reverse transcription of total RNAs isolated from cells treated with PPE revealed that the mRNA expressions of IL-1β, IL-6, TNFα, and NO synthase (NOS)-2 were reduced. The necessary concentration of PPE prepared by enzymatic digestion to mediate anti-inflammatory effects compared with the reported value of that extracted by phosphate buffered saline without digestion was proportional to the amount of extracted materials from the same amount of placenta (about 10-fold). This suggests that the molecules responsible for the anti-inflammatory activity exists in the placenta and can be extracted by phosphate buffered saline, and thus might survive enzymatic digestion.