Objective: To study the anti-inflammatory and analgesic effects of Ski protein overexpression on mice induced by acetic acid.? Methods: Eight-week-old male ICR mice were selected and given 0.7% acetic acid solution (0.1ml/10g body weight) to induce writhing reaction.Experimental mice were divided into Sham group, acetic acid group, acetic acid + ibuprofen group, acetic acid +Ad-EGFP group, acetic acid +Ad-ski-1 group and acetic acid +Ad-ski-2 group, with 10 mice in each group. Observe and record the incubation period for the first appearance of twisting, while also recording the number of twisting times within 15 minutes. Obtain small intestine tissue, identify adenovirus transfection effect, detect protein expression levels of pro-inflammatory factors and pain biomarkers, and further analyze the protein expression of NF-kB p65 and its binding with Ski protein. Results:Ski protein was successfully overexpressed in small intestine after intraperitoneal injection of Ad-ski adenovirus. Overexpressed Ski protein prolonged the incubation period of writhing and decreased the frequency of writhing reaction, which was comparable to ibuprofen (P > 0.05). Compared with acetic acid group, overexpressed Ski protein significantly inhibited the protein expression of pro-inflammatory factors and pain biomarkers (*P < 0.05,**P < 0.01). Moreover, NF-kB p65 forms complexes with Ski. Conclusion: Overexpression of Ski protein has anti-inflammatory and analgesic effects on acetic acid induced inflammatory pain by inhibiting the expression of inflammatory factors and pain biomarkers, and is related to Ski regulation of the NF-kB signaling pathway.