BACKGROUND: Diabetes mellitus (DM) involves metabolic changes that can negatively influence periodontal tissues, resulting in impaired periodontal repair. There is a lack of information about the outcomes of regenerative approaches under the influence of DM. Enamel matrix derivatives (EMDs) have been used in periodontal regenerative procedures, resulting in improvement of clinical parameters. Thus, the aim of this histomorphometric study is to evaluate the healing of periodontal defects after treatment with EMD under the influence of DM. METHODS: Twenty Wistar rats were randomly assigned to two groups: group 1 (G1): DM was induced with a single intraperitoneal injection of streptozotocin (STZ) (n = 10); group 2 (G2): rats were not exposed to STZ (n = 10). Seven days after DM induction, bilateral fenestration defects were created at the buccal aspect of the first mandibular molar. After the surgeries, the defects of each animal were randomly assigned to two subgroups: non-treated (control) and treated with EMD. The animals were euthanized 21 days later, and the percentage of defect fill (DF), newly formed bone density (BD), and new cementum formation (NCF) were histometrically assessed. The number of osteoclasts was determined by tartrate-resistant acid phosphatase. Weight and blood glucose were also analyzed. Mann-Whitney U test was used for comparison among groups and Wilcoxon test for comparison between the start and end times (weight and blood glucose) and between treatments (NCF and number of osteoclasts). One-way analysis of variance was used to assess DF and BD. Tukey test was used when the analysis of variance test detected significant differences (α = 5%). RESULTS: G1 (DM) showed less DF and BD compared with G2. EMD provided an increased DF in both groups and enhanced BD and NCF only in G2. The number of TRAP-positive osteoclasts was significantly higher in EMD-treated sites of G1. CONCLUSIONS: DM may produce a significant detrimental effect on BD. EMD may provide greater DF under diabetic or normal conditions; however, it may not significantly increase NCF in animals with DM.