n-3 polyunsaturated fatty acids (PUFAs) can improve the function of the intestinal barrier after damage from ischemia-reperfusion or hemorrhagic shock resuscitation (HSR). However, the effects of n-3 PUFAs on intestinal microbiota and the innate immunity of the intestinal mucosa after HSR remain unclear. In the present study, 40 C57BL/6J mice were randomly assigned to five groups: control, sham, HSR, HSR + n-3 PUFAs and HSR + n-6 PUFAs. Mice were sacrificed 12 h after HSR. Liver, spleen, mesenteric lymph nodes and terminal ileal tissues were collected. Intestinal mucosae were scraped aseptically. Compared with the HSR group, the number of goblet cells increased, expression of mucin 2 was restored and disturbed intestinal microbiota were partly stabilized in the PUFA-administered groups, indicating that both n-3 and n-6 PUFAs reduced overproliferation of Gammaproteobacteria while promoting the growth of Bacteroidetes. Notably, n-3 PUFAs had an advantage over n-6 PUFAs in improving ileal tissue levels of lysozyme after HSR. Thus, PUFAs, especially n-3 PUFAs, partly improved the innate immunity of intestinal mucosa in mice after HSR. These findings suggest a clinical rationale for providing n-3 PUFAs to patients recovering from ischemia-reperfusion.