We tested the hypothesis that improper myonuclei arrangement and morphology are involved in diabetes-induced myofiber atrophy and whether and how high-intensity interval training (HIIT) affects these impairments in isolated skeletal muscle myofibers. STZ-induced diabetes decreased muscle fiber cross-sectional area (CSA) mediated by reduced myonuclear number, enhanced nuclear apoptotic, and failed nuclear accretion from satellite cells. STZ-induced muscle atrophy was accompanied by improper nuclear positioning (sinus of the maximum diameter angles and distance between adjacent myonuclei) and morphology (maximum diameter, area, and volume of the nuclei), which was mediated by suppressed expression of proteins involved in nuclear positioning including KIF5B, dynein, and Nesprin1. Disturbing nuclear positioning by inhibition of Kinsein1 activity reduced CSA to a greater extent than in diabetes alone, suggesting STZ-induced muscle atrophy is mediated by changes in nuclear positioning. HIIT alleviated the STZ-induced decline in muscle CSA and myonuclei per fiber by restoring myonuclear morphometry impairments and improper nuclear positioning to the normal level. HIIT-induced increase in muscle CSA deterred by inhibition of Kinesin1 activity, suggesting its effect is mediated by proper nuclear positioning. These findings suggest that normal nuclear positioning are required for the changes in fiber size properties associated with HIIT in diabetic skeletal muscle fibers.