Background: In the battle against Crohn’s disease, autophagy stimulation is a promising therapeutic option—one both new and newly rediscovered. In experimental models, docosahexaenoic acid (DHA)—a long-chain polyunsaturated fatty acid—has been demonstrated to be useful in the treatment of inflammatory bowel disease through inhibition of the nuclear factor-κB pathway. However, the impact of DHA on autophagy in the colon remains unclear. Methods: Mice were divided into 3 groups: wild type (placebo), the interleukin 10 knockout group (IL-10−/−, placebo), and the DHA group (IL-10−/−, DHA). DHA was administered to IL-10−/− mice by gavage at a dosage of 35.5 mg/kg/d for 2 weeks. The severity of colitis, expression of proinflammatory cytokines, expression/distribution of LC3B, and mTOR signaling pathway were evaluated in the proximal colon tissues collected from all mice at the end of the experiment. Results: DHA administration ameliorated experimental colitis in the IL-10−/− mice, as demonstrated by decreased proinflammatory cytokines (TNF-α and IFN-γ), reduced infiltration of inflammatory cells, and lowered histologic scores of the proximal colon mucosa. Moreover, in the DHA-treated mice, enhanced autophagy was observed to be associated with (1) increased expression and restoration of the distribution integrity of LC3B in the colon and (2) inhibition of the mTOR signaling pathway. Conclusion: This study showed that DHA therapy could attenuate experimental chronic colitis in IL-10−/− mice by triggering autophagy via inhibition of the mTOR pathway.