Background: This study evaluated the effect of different dietary ω-6/ω-3 polyunsaturated fatty acid (PUFA) ratios on modulating helper T (Th) and regulatory T (Treg) lymphocytes in mice with dextran sulfate sodium (DSS)–induced colitis. Methods: There were 3 control and 3 colitis groups. Mice were fed for 24 days with diets with soybean oil (S), a mixture of soybean oil and low fish oil content (LF), or high fish oil content (HF). The ratio of ω-6/ω-3 PUFA in the LF diet was 4:1, and that in the HF diet was 2:1. The control groups drank distilled water while colitis groups were provided 2% DSS in drinking water during days 15–19. All mice drank distilled water from days 20–24 for recovery and were sacrificed on day 25. Results: Colitis resulted in higher blood Th1, Th2, and Th17 and lower Treg percentages. Also, plasma haptoglobin and proinflammatory chemokines were elevated in colon lavage fluid. Colitic groups with fish oil had lower inflammatory mediators in the plasma and colon lavage fluid. Furthermore, the percentages of blood Th1, Th2, and Th17 cells were lower, whereas Treg cell percentages were higher than those in the soybean oil group. The colitis group with an ω-6/ω-3 PUFA ratio of 2:1 had more pronounced effects than the group with a ratio of 4:1. Conclusions: Diets with an ω-6/ω-3 PUFA ratio of 2:1 or 4:1 regulate the Th/Treg balance and attenuate inflammatory mediator production in colitis. Compared with the ω-6/ω-3 PUFA ratio of 4:1, the ratio of 2:1 was more effective in reducing inflammatory reactions in DSS-induced colitis.