Mitsugumin 53 (MG53) is an E3 ligase that induces insulin receptor substrate-1 (IRS-1) ubiquitination and degradation in skeletal muscle. We previously demonstrated that the pharmaceutical disruption of the MG53-IRS-1 interaction improves insulin sensitivity by abrogating IRS-1 ubiquitination and increasing IRS-1 levels in C2C12 myotubes. Here, we developed a novel MG53-IRS-1 interaction disruptor (MID-00935) that ameliorates insulin resistance in diet-induced obese (DIO) mice. MID-00935 disrupted the molecular interaction of MG53 and IRS-1, abrogated MG53-induced IRS-1 ubiquitination and degradation and improved insulin signaling in C2C12 myotubes. Oral administration of MID-00935 increased insulin-induced IRS-1, Akt, and Erk phosphorylation via increasing IRS-1 levels in the skeletal muscle of DIO mice. In DIO mice, MID-00935 treatment lowered fasting blood glucose levels and improved glucose disposal in glucose and insulin tolerance tests. These results suggest that MID-00935 may be a potential muscle-targeting drug candidate for treating insulin resistance.