Skeletal muscle has a central role in maintaining metabolic homeostasis. 17α-estradiol (17α-E2), a naturally-occurring non-feminizing diastereomer of 17β-estradiol that demonstrates efficacy for improving metabolic outcomes in male, but not female, mice. Despite several lines of evidence showing that 17α-E2 treatment improves metabolic parameters in middle-aged obese and old male mice through effects in brain, liver, and white adipose tissue little is known about how 17α-E2 alters skeletal muscle metabolism, and what role this may play in mitigating metabolic declines. Therefore, this study aimed to determine if 17α-E2 treatment improves metabolic outcomes in skeletal muscle from obese male and female mice following chronic high fat diet (HFD) administration. We hypothesized that male, but not female, mice, would benefit from 17α-E2 treatment during HFD. To test this hypothesis, we used a multi-omics approach to determine changes in lipotoxic lipid intermediates, metabolites, and proteins related to metabolic homeostasis. In male mice, we show that 17α-E2 alleviates HFD-induced metabolic detriments of skeletal muscle by reducing the accumulation of diacylglycerol (DAGs) and ceramides, inflammatory cytokine levels, and reduced the abundance of most of the proteins related to lipolysis and beta-oxidation. In contrast to males, 17α-E2 treatment in female mice had little effect on the DAGs and ceramides content, muscle inflammatory cytokine levels, or changes to the relative abundance of proteins involved in beta-oxidation. These data support to the growing evidence that 17α-E2 treatment could be beneficial for overall metabolic health in male mammals.