Enteromorpha prolifera (EP) is a green alga that causes green bloom worldwide. This study aimed to isolate and identify n-3 polyunsaturated fatty acids (PUFAs) from EP oil obtained via supercritical fluid extraction (SFE) and to explore its preventive effects against dextran sodium sulfate (DSS)-induced ulcerative colitis in C57BL/6J mice. In EP oil, we found the novel n-3 polyunsaturated fatty acid C16:4n-3 and two unusual fatty acids C18:4n-3 and C16:3n-3, using GC-MS. The administration of EP oil reduced histopathological of symptoms colitis and the shortening of the colon length. Pro-inflammatory cytokines of IL-6 and TNF-α in serum of EP oil treatment were lower than DSS treatment (by 37.63% and 83.52%), and IL-6 gene expression in the colon was lower in than DSS group by 48.28%, and IL-10 in serum was higher than DSS group by 2.88-fold. Furthermore, the protein expression of p-STAT3 by the EP oil treatment was significantly reduced compared with DSS treatment group by 73.61%. Lipidomics study suggested that phosphatidylcholine and phosphatidylethanolamine were positively associated with the anti-inflammatory cytokine IL-10, while cholesteryl ester and sphingomyelin were negatively related to inflammation cytokines in the EP oil group. The present results indicated that EP oil rich in n-3 PUFA contains a novel fatty acid C16:4n-3, as well as two uncommon fatty acids C18:4n-3 and C16:3n-3. EP oil could prevent DSS-induced ulcerative colitis by regulating the JAK/STAT pathway and lipid metabolism.