Objective: Diabetes mellitus is closely associated with mitochondrial dysfunction, which disrupts cellular energy metabolism and perturbs hormonal homeostasis. Humanin (HN), a 24-amino acid peptide encoded within the mitochondrial genome, has attracted considerable attention due to its cytoprotective and metabolic regulatory properties. Despite its recognized biological potential, the role of HN in coordinating key metabolic hormone networks under diabetic conditions remains poorly understood. This study aimed to investigate the integrative effects of repeated humanin administration on key metabolic hormones leptin, ghrelin, irisin, and asprosin and its potential role in restoring hormonal homeostasis in a streptozotocin (STZ)-induced diabetic mouse model. Materials and Methods: Forty male mice were randomly assigned to four groups (n = 10 for each group): control, HN (4 mg/kg/day), STZ (150 mg/kg), and STZ + HN. Humanin was administered intraperitoneally for 15 consecutive days. Serum levels of leptin, asprosin, irisin, and ghrelin were measured using enzyme-linked immunosorbent assay (ELISA), and data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Results: STZ-induced diabetes markedly disrupted metabolic hormone balance, as indicated by decreased leptin and irisin levels and increased asprosin concentrations. Repeated HN treatment effectively restored leptin levels and suppressed asprosin concentrations, while irisin levels showed a relative increase compared to the STZ animals. In addition, ghrelin levels were significantly elevated in HN-treated diabetic mice compared to untreated STZ animals. Conclusions: These findings indicate that humanin exerts an integrative, multi-hormonal regulatory effect, supporting the restoration of metabolic and endocrine homeostasis under diabetic conditions.