Periodontitis is an oral-cavity inflammatory disease and is the principal cause associated with tooth loss. Matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) are important proteases involved in periodontal tissue destruction. The omega-3 polyunsaturated fatty acids (ω-3 PUFA) have been demonstrated to possess immunoregulatory properties in periodontitis. The aim of the study was to investigate the effects of ω-3 PUFA on inflammation and on the expression of MMP-2 and -9 in a murine periodontitis model. Twenty-four male C57BL/6 mice were divided into control mice (Control), control mice treated with ω-3 PUFA (O3), mice with periodontitis (P), and mice with periodontitis treated with ω-3 PUFA (P + O3). ω-3 PUFA were administered orally once a day for 70 days. Periodontitis in mice was induced by Porphyromonas gingivalis-infected ligature placement around the second maxillary molar. The mice were sacrificed, and blood and maxillary samples were collected. Flow cytometry was used to quantify tumor necrosis factor-alpha (TNFα), interleukin (IL)-2, IL-4, IL-5, and interferon-gamma. Histologic analysis and immunohistochemistry for MMP-2 and -9 were performed. The data were statistically evaluated using analysis of variance (ANOVA) and the Tukey post hoc test. Histological analysis showed that ω-3 PUFA supplementation prevented inflammation and tissue destruction and revealed that bone destruction was more extensive in the P group than in the P + O3 group (p < 0.05). Also, it decreased the serum expressions of TNFα and IL-2 and the tissue expression of MMP-2 and -9 in the periodontitis-induced model (p < 0.05). ω-3 PUFA supplementation prevented alveolar bone loss and periodontal destruction, probably by decreasing the expression of MMP-2 and MMP-9 and its immunoregulatory properties.