There is increasing evidence that multiple proteins involved in key regulatory processes in mitochondria are phosphorylated in mammalian tissues. Insulin regulates glucose metabolism by phosphorylation-dependent signaling and has been shown to stimulate ATP synthesis in human skeletal muscle. Here, we investigated the effect of insulin on the phosphorylation of mitochondrial proteins in human skeletal muscle in vivo. Using a combination of TiO(2) phosphopeptide-enrichment, HILIC fractionation, and LC-MS/MS, we compared the phosphoproteomes of isolated mitochondria from skeletal muscle samples obtained from healthy individuals before and after 4 h of insulin infusion. In total, we identified 207 phosphorylation sites in 95 mitochondrial proteins. Of these phosphorylation sites, 45% were identified in both basal and insulin-stimulated samples. Insulin caused a 2-fold increase in the number of different mitochondrial phosphopeptides (87 ± 7 vs 40 ± 7, p = 0.015) and phosphoproteins (46 ± 2 vs 26 ± 3, p = 0.005) identified in each mitochondrial preparation. Almost half of the mitochondrial phosphorylation sites (n = 94) were exclusively identified in the insulin-stimulated state and included the majority of novel sites. Phosphorylation sites detected more often or exclusively in insulin-stimulated samples include multiple sites in mitochondrial proteins involved in oxidative phosphorylation, tricarboxylic acid cycle, and fatty acid metabolism, as well as several components of the newly defined mitochondrial inner membrane organizing system (MINOS). In conclusion, the present study demonstrates that insulin increases the phosphorylation of several mitochondrial proteins in human skeletal muscle in vivo and provides a first step in the understanding of how insulin potentially regulates mitochondrial processes by phosphorylation-dependent mechanisms.