In the United States, over three million adults suffer from inflammatory bowel disease (IBD). The gut microbiome, host immune response, and nutrient-microbial interactions are known to play a role in IBD. The relationship between dairy and IBD is controversial; thus, the objectives of this study were to identify how milk polar lipids (MPLs) and anhydrous milk fat affect colitis disease activity, the colonic transcriptome, and the gut microbiome in a mouse model of chemical-induced colitis. Male and female C57BL/6J mice (n = 120) were randomized into either a low (5% w/w) milk fat or a high (21% w/w) milk fat diet supplemented with either 0%, 1%, or 2% w/w of MPLs for three weeks (n = 10/group/sex). Afterwards, colitis was induced using 1% dextran sodium sulfate in drinking water for five days (colitis induction) and then switched to regular water for five days (colitis recovery). Mice fed added MPLs were protected against colitis when fed a high-fat diet, while added MPLs during low-fat diet attenuated disease activity during the colitis induction period yet promoted colitis and inflammation in male mice during the recovery period. Dietary fat content can alter colitis and influence the anti-inflammatory effect of milk polar lipids.