This study investigates the mitochondrial morphology of intestinal stem cells (ISCs) in different regions of the Drosophila midgut—functionally similar to the human intestine—across sexes and under a high-sugar diet (HSD). The Drosophila midgut is divided into five regions from anterior to posterior (R1 to R5), each containing well-characterized ISCs. Our results reveal slight regional differences in ISC mitochondrial morphology. Interestingly, in the R1 region, responsible for food digestion, female ISC mitochondria are longer and larger than those in males. Conversely, in the R5 region, associated with waste excretion, male ISC mitochondria are longer and larger than those in females. More strikingly, HSD reduces mitochondrial length and size in female ISCs but increases them in male ISCs. These findings suggest that mitochondrial morphology is shaped by both sex and diet, highlighting the potential for dietary interventions to alter mitochondrial function for treating intestinal metabolic disorders.