The Portulaca oleracea L. oligopeptides are seldom explored because they are often present in a complex matrix. In the current study, eleven novel Portulaca oleracea L. oligopeptides (POPs) were isolated and their mino acid sequence identified. Further, the anti-inflammatory potency was explored in lipopolysaccharide (LPS)-induced RAW264.7 cells. Results showed that POP-1∼[EHGEYE] possessed excellent anti-inflammatory potency by attenuating the pro-inflammatory cytokine expression (TNF-α, NO, IL-1β); inhibiting iNOS and COX-2 expressions and regulating the MAPK, PI3K/Akt and NF-κB signaling pathways. This may be an important molecular mechanism of POPs in anti-inflammatory damage.