Abstract Body: Background: Diabetic cardiomyopathy (DCM) is the leading cause of mortality in diabetic patients, accounting for about 80% of the death. Mitochondria and endoplasmic reticulum (ER) play a critical role in cardiomyocyte function, yet their interaction and function in diabetic hearts remains unclear. Hypothesis: We hypothesize that mitochondria and ER stress response are essential in maintaining cardiac function and preventing cardiac remodeling during early diabetic insult in the heart. Methods: Utilizing 6-month-old male and female C57BL/6 mice, we divided them into groups receiving a standard chow diet (N=8) and a 60% high-fat diet (HFD, N=8) for four months. Post-diet, we conducted glucose tolerance tests to verify hyperglycemia. We analyzed heart samples for mitochondrial stress (LonP1, ClpX, ClpP, Afg3l2, spg7, GRP75) and ER stress (XbP1, IRE1a, CHOP, PDI, Hspa5, Dnajb9, Hsp90) markers, along with mitochondrial biogenesis (mtDNA, TFAM, Mt-ND4, and Mt COII levels), and whole heart homogenate oxidative potential by electron paramagnetic resonance (EPR) spin probe (CMH). Between the groups, a p-value < 0.05 was considered significant calculated by one-way ANOVA. Results: HFD significantly increased body weight (p<0.0001) and fasting glucose levels (p<0.0001), confirming hyperglycemia. Among the mitochondrial stress response markers, LonP1 was significantly (p<0.05) elevated in HFD hearts compared to the control. However, the ER stress-associated transcription factors, CHOP and XbP1, were significantly upregulated (p<0.05) in female HFD hearts compared to the control and male groups. Further, EPR analysis showed a significant (p<0.0001) four- and two-fold increase in oxidative potential in the heart homogenate of female and male HFD groups, respectively. However, mitochondrial biogenesis was notably higher, observed only in HFD females compared to other groups. Conclusion: Our study indicates that pre-diabetic hyperglycemia enhances gender-specific mitochondrial biogenesis and triggers mitochondrial and ER stress responses, highlighting the importance of mitochondrial-ER interaction in early DCM and suggesting potential therapeutic avenues.