Banana fruits are harvested and then undergo rapid ripening and senescence, sharply limiting their shelf-life and marketability. Myo-inositol (MI) is an important regulator in ethylene production and reactive oxygen species (ROS) accumulation; however, its involvement in the postharvest ripening process of banana remains to be determined. This study found that postharvest application of MI could efficiently delay the fruit ripening and extend the time in which the luster, color, and hardness were maintained in two cultivars with contrasting storage characteristics, storable ‘Brazil’ and unstorable ‘Fenza No. 1’, when stored at room temperature (23 °C ± 2 °C). Moreover, physiological, metabolic, and gene expression analyses indicated that MI application improved MI metabolism and postponed ethylene biosynthesis and cell wall loosening. The decrease in ethylene production was associated with a reduction in the expression of ACS1 and ACO1 genes. MI treatment decreased the expressions of PL1/2, PG, and EXP1/7/8, which may account for the delay in softening. In addition, the application of MI could alleviate ROS-mediated senescence and cell membrane damage by promoting the activities of SOD, POD, and anti-O₂⁻ and decreasing PPO activity. This study shed light on the function of MI in regulating the postharvest ripening and senescence of bananas and provided an efficient strategy for extending shelf-life and reduce losses.