<p>This study aimed to investigate autophagy and its associated mechanisms in periodontal ligament stem cells (PDLSCs) within the inflammatory microenvironment of type 2 diabetes mellitus (T2DM) and periodontitis. Periodontal ligament tissues were obtained from healthy individuals, individuals with T2DM, individuals with chronic periodontitis, and individuals with both T2DM and periodontitis. PDLSCs were isolated, cultured, and treated with the autophagy inhibitor 3-methyladenine (3-MA) and the autophagy activator rapamycin (Rapa). Cell proliferative capacity was evaluated, autophagic activity and organelle damage were assessed using transmission electron microscopy, and the relative expression levels of autophagy-related genes (<i>Beclin-1</i>,<i> LC3 II</i>,<i> P62</i>) were measured using real-time quantitative PCR. Compared to PDLSCs derived from healthy individuals, those from individuals with chronic periodontitis or T2DM exhibited no significant morphological differences but demonstrated reduced proliferative capacity. Treatment with 3-MA and Rapa did not significantly alter proliferative capacity across groups. PDLSCs from individuals with chronic periodontitis and T2DM displayed increased autophagosome formation, more severe organelle damage, and upregulated expression of autophagy-related genes <i>Beclin-1</i> and <i>LC3 II</i>, while P62 expression was downregulated, compared to PDLSCs from healthy individuals. PDLSCs from individuals with T2DM and periodontitis exhibit excessive autophagy and organelle damage. Autophagy dysregulation in PDLSCs within a diabetic and inflammatory microenvironment may contribute to the severity of periodontal destruction observed in individuals with T2DM.</p>