Abstract: This study was designed to investigate the synergistic analgesic effect between choline (Cho) and acetaminophen (Ace). Mice were treated with 0.6% acetic acid solution by intraperitoneal injection to build acetate writhing model. The KM mice were randomly divided into four groups:control group (n=10), Cho group (n=50), Ace group (n=50), combination group (Cho+Ace group, n=40), then the writhing times were counted respectively. OriginPro8.5 was used to calculate ED 50. The isobolographic analysis was used to test the interaction of Cho and Ace. To explore the mechanism, forty KM mice were randomly divided into control group, Cho group, Ace group and Cho + Ace group. Blood was collected for detection of TNF-α, IL-6, PGE₂ and NF-κB content using ELISA kits. The result ED 50 was calculated as followings. ED⁵⁰ of Cho and Ace was 19.47 mg·kg^-1 and 20.56 mg·kg^-1. The concentrations were 2.94 mg·kg^-1 for Cho and 3.15 mg·kg^-1 for Ace in the combination test. The levels of TNF-α, IL-6, PGE₂ and NF-κB in Cho group and Ace group were lower than those in the control group (P< 0.05). Compared to the Cho group and Ace group, the levels of TNF-α, IL-6, PGE₂, NF-κB in Cho + Ace group were reduced further (P< 0.05). The results revealed that Cho and Ace have synergistic analgesic effects, which may associate with inhibition of the NF-κB signaling pathway.