Physical inactivity/disuse results in skeletal muscle metabolic disruption including insulin resistance and mitochondrial dysfunction. The role of the toll-like receptor 4 (TLR4) signaling pathway in contributing to metabolic decline with muscle disuse is unknown. Therefore, our goal was to determine if TLR4 is an underlying mechanism of insulin resistance, mitochondrial dysfunction, and skeletal muscle ceramide accumulation following muscle disuse in mice. To address this hypothesis, we subjected (n=6-8/group) male WT and TLR4^-/- mice to 2-weeks of hindlimb unloading (HU) while a second group of mice served as ambulatory wild-type controls (WT CON, TLR4^-/- CON). Mice were assessed for insulin resistance (HOMA-IR, glucose tolerance) and hindlimb muscles (soleus and gastrocnemius) were used to assess muscle sphingolipid abundance, mitochondrial respiration (respiratory control ratio (RCR)), and NFB signaling. The primary finding was that HU resulted in insulin resistance, increased total ceramides, specifically Cer18:0 and Cer20:0, and decreased skeletal muscle mitochondrial respiration. Importantly, TLR4^-/- HU mice were protected from insulin resistance and altered NFB signaling and were partly resistant to muscle atrophy, ceramide accumulation and decreased RCR. Skeletal muscle ceramides and RCR were correlated with insulin resistance. We conclude that TLR4 is an upstream regulator of insulin sensitivity while partly upregulating muscle ceramides and worsening mitochondrial respiration during 2-weeks of HU.