Objective To study the anti-inflammatory and analgesic effects of Ski protein overexpression on writing in mice induced by acetic acid. Methods Eight-week-old male ICR mice were administered 0. 7% acetic acid solution (0. 1 mL/ 10 g) to induce a writhing reaction. The mice were divided into sham, acetic acid, acetic acid + ibuprofen, acetic acid + ad-EGFP, acetic acid + ad-ski-1, acetic acid + ad-ski-2, and acetic acid + sulfasalazine groups ( n = 10 mice per group). The time to the first appearance of twisting and the number of twists within 15 min were recorded. Small intestine tissues were removed to identify the effect of adenovirus transfection and to detect protein expression levels of proinflammatory factors and pain biomarkers and protein expression of nuclear factor (NF)-κB 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 delayed the start and decreased the frequency of writhing, comparable to ibuprofen (P> 0. 05). Groups in which ski protein was overexpressed showed significantly inhibited protein expression of proinflammatory factors and pain biomarkers compared with the acetic acid group (P< 0. 05). Moreover, NF-κB p65 formed complexes with Ski. Conclusions Overexpression of Ski protein has anti-inflammatory and analgesic effects on acetic acidinduced inflammatory pain by inhibiting the expression of inflammatory factors and pain biomarkers, via regulation of the NF-κB signaling pathway.