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Endurance training enhances the capacity for fat oxidation during exercise due to increased utilization of intramuscular lipid (IMCL). This study quantitatively investigated the impact of exercise training status on muscle fiber type-specific abundance of regulatory proteins involved in IMCL utilization. Endurance-trained [ n = 7 subjects, peak oxygen consumption (V̇o 2peak ) 62.6 ± 4.1 (SD) mL·min -1 ·kg -1 ] and non-endurance-trained ( n = 8 subjects, V̇o 2peak 44.9 ± 5.3 mL·min -1 ·kg -1 ) young men completed an incremental exercise test to determine maximal fat oxidation (MFO) and maximal oxygen uptake. Fiber type-specific IMCL content and protein abundance were assessed with immunofluorescence microscopy and immunoblot analysis of pooled single muscle fibers and whole muscle. Endurance-trained individuals displayed a higher MFO rate (0.45 ± 0.15 vs. 0.19 ± 0.07 g/min, P < 0.05), a greater proportion of type I muscle fibers, and higher IMCL content compared with untrained individuals ( P < 0.05). Adipose triglyceride lipase, hormone-sensitive lipase, perilipin 2, perilipin 5, and hydroxyacyl-coenzyme A dehydrogenase abundances were ~2-3-fold higher in type I muscle fibers compared with type IIa fibers ( P < 0.05). Correspondingly, these lipid proteins and oxidative enzymes were higher in endurance-trained individuals when assessed in whole muscle. MFO rate was strongly related to the proportion of type I fibers ( R  = 0.81, P < 0.01). The abundance of proteins involved in the regulation of IMCL storage and oxidation is highly muscle fiber type specific. The increased capacity for fat oxidation in endurance-trained individuals corresponded with increased IMCL content and elevated abundance of lipolytic and oxidative enzymes in combination with a greater proportion of type I muscle fibers. NEW & NOTEWORTHY We have utilized contemporary techniques to compare the fiber type-specific characteristics of skeletal muscle from endurance-trained athletes and untrained individuals. We show that type I muscle fibers have a coordinated upregulation of proteins controlling intramuscular lipid storage, mobilization, and oxidation. Furthermore, the enhanced capacity for intramuscular lipid storage and utilization in endurance-trained individuals is related to the increased expression of lipid regulatory proteins combined with a greater proportion of type I muscle fibers.