Periodontitis is a chronic inflammatory disease that affects the tooth-supporting tissues. This study evaluated the anti-inflammatory and antiresorptive effects of milk kefir (MK) on periodontitis in rats. Micro-Raman spectroscopy was performed on MK at different fermentation times to verify the presence of Lactobacillus kefiri. From these results, Wistar rats were divided into the following groups: C (Control); EP (experimental periodontitis); K1 (animals that received MK with one day of fermentation); K1+EP; K4 (animals without EP using MK with four days of fermentation) and K4+EP. MK was administered 28 days before EP induction and during the disease development period (11 days). On day 28, in the EP groups, periodontitis was induced. The animals were euthanized on day 39. The hemimaxillae were removed and the following parameters were evaluated: micro-Raman analysis of the presence of inflammation; histomorphometric analysis to quantify alveolar bone loss and immunohistochemistry for IL-6, TNF-α, IL-Iβ and IL-10 in the periodontal ligament. Micro-Raman analysis showed that four days fermentation MK has a higher intensity spectrum of L. kefiri. Furthermore, the administration of this probiotic reduced the intensity of the inflammation spectrum when compared to one day fermentation MK. It was observed that the animals from the K4+EP group showed significant reduction of alveolar bone loss, as well as a lower IL-6, TNF-α and IL-Iβ immunoexpression and a higher IL-10 immunoexpression, when compared to EP groups. We conclude that MK has anti-inflammatory and antiresorptive effects on periodontitis in rats and that these effects are fermentation time dependent.