Dysbiotic subgingival biofilms initiate periodontitis, while the consequential destruction of periodontal tissues results from a dysregulated local immune response. Interstitial CD4 + T cells play a crucial role in orchestrating periodontal inflammation. Upon activation, CD4 + T cells express CD69 receptors, which can influence their migration patterns, phenotype, and function during inflammation. Here, we report that in the absence of CD69, memory CD4 + T cells (mCD4 + T cells) derived from gingival and cervical lymph nodes (cLNs) display an increased proinflammatory phenotype. Following in vitro activation, negative-selected mCD4 + T cells from cLNs of CD69 KO mice showed enhanced expression of interleukin (IL)–17A ( P = 0.0043) and interferon-γ ( P = 0.0479). Although comparable to untreated wild-type (WT) mice in the absence of disease, CD69-deficient mice showed augmented alveolar bone loss and a greater interstitial inflammatory cell infiltrate after 7 d of ligature-induced experimental periodontitis. Furthermore, gingival CD4 + T cells derived from mice lacking CD69 produced significantly higher levels of IL-17A compared with WT animals. 16S rRNA gene sequencing and bioinformatics analyses of the subgingival microbiota associated with ligatures indicated that the absence of CD69 in the host significantly shaped the composition of the periodontitis-associated biofilm. Therefore, our data suggest that CD69 receptors play a regulatory role in both the cellular and microbial microenvironments associated with periodontitis.