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A novel homogeneous polysaccharide, designated as PLPN-2, was screened from the dried root of Paeonia lactiflora Pall. using an activity-oriented approach, demonstrating remarkable anti-inflammatory properties. Structural analysis revealed that PLPN-2 is an α-glucan comprising an α-(1 → 4)-glucan backbone with α-Glcp-(1→/α-Glcp-(1 → 6)-β-Glcp-(1→ linked to the backbone at C-6. PLPN-2 exhibited efficacy in alleviating inflammatory symptoms associated with DSS-induced colitis in mice and inhibiting the release of pro-inflammatory elements including IL-6, TNF-α, IL-1β from RAW264.7 macrophages promoted by LPS. Moreover, PLPN-2 demonstrated ability to impede the nuclear translocation of the p65 protein and reduce the phosphorylation levels of JNK, ERK1/2, p38, p65, and IκBα. The findings suggest that PLPN-2 exhibits potential as an anti-inflammatory agent through the inhibition of the MAPK and NF-κB signaling pathways. Therefore, PLPN-2 shows promising prospects as a functional agent for relieving inflammation-related disorders.