A long-term high-fat diet (HFD) is one of the high-risk factors for intestinal barrier damage. Docosahexaenoic acid-enriched phosphatidylserine (DHA-PS) has multiple biological activities, while its protective effect on HFD-caused jejunum injury remains unknown. Thus, the present study investigated the protective effect of DHA-PS on HFD-induced jejunum injury in mice. Our results showed that DHA-PS (100 mg per kg per d) could protect against HFD-caused jejunum injury by decreasing the levels of inflammatory factors such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) in the serum and jejunum tissues, with histological analysis confirming this injury amelioration. Additionally, DHA-PS alleviated the HFD-caused oxidative stress by decreasing malondialdehyde (MDA) and increasing total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) levels in the jejunum. Moreover, DHA-PS significantly increased the expression of tight junction proteins (ZO-1, occludin, and claudin-4) in the jejunum, and modulated the HFD-induced gut microbiota disorder by decreasing the Firmicutes and Bacteroidetes ratio, and reducing the relative abundance of Lachnoclostridium, Coriobacteriaceae, Desulfovibrionaceae, and Helicobacter, while increasing the relative abundance of Lachnospiraceae_NK4A136_group, Alistipes, norank_f__Muribaculaceae, and Bacteroides. Overall, these results support that DHA-PS can alleviate the HFD-caused jejunum injury.