A long-term Western diet high in trans-fatty acids (TFAs) is associated with an increased risk of obesity; however, whether and how such a diet exacerbates obesity-related muscle atrophy remains unclear. To investigate the effects of TFA on muscle atrophy, serum bile acid, and muscle mitochondrial function in the context of obesity, C57BL/6 mice were initially fed a high-fat, high-cholesterol (HFHC) diet for 8 weeks to establish an obesity model. Subsequently, mice were subjected to a 20-week TFA intervention, wherein soybean oil was replaced with an isocaloric amount of partially hydrogenated soybean oil to induce muscle atrophy (n = 8 per group). Skeletal muscle mass, insulin resistance, serum bile acid composition, and skeletal muscle transcriptomic alterations were assessed. The results showed that insulin resistance was exacerbated in obese mice following TFA intervention (P < 0.05), and muscle fiber cross-sectional area was significantly reduced (P < 0.05). Moreover, in obese mice, serum levels of deoxycholic acid and taurodeoxycholic acid increased significantly by 238.99% and 281.38%, respectively. Compared with the HFHC group, the expression levels of mitochondrial electron transport chain-related genes, including Ndufaf8, Uqcrh, and mt-Co3, were significantly decreased in the TFA group (P < 0.05). Thus, muscle atrophy may be exacerbated by TFAs in obese mice, potentially through a mechanism involving increased hydrophobicity of serum bile acid, which disrupts mitochondrial electron transport chain function in muscle. Collectively, these findings suggest that TFAs may serve as a dietary risk factor exacerbating muscle atrophy in the context of obesity, indicating that restricting dietary TFA intake holds promise as an effective strategy for ameliorating obesity-related muscle atrophy.