This study explored key proteins involved in fat metabolism that may associate with peak fat oxidation (PFO) and account for sexual dimorphism in exercise fuel metabolism. Thirty-six healthy adults [15 females; age 40 (11); V̇O2peak 42.5 (9.5) mLkg BM-1min-1; means±SD] completed two exercise tests to determine PFO via indirect calorimetry. Resting adipose tissue and/or skeletal muscle biopsies were obtained to determine the protein content of adipose tissue PLIN1, CGI-58, HSL, ATGL, ACSL1, CPT1b and oestrogen receptor α (ERα), and skeletal muscle FABPpm, ATGL, ACSL1, CTP1b and ERα. Moderate strength correlations were found between PFO (mgkg FFM-1min-1) and the protein content of ATGL [rs=0.41 (0.03–0.68), P<0.05] and CPT1b [rs=0.45 (0.09–0.71), P<0.05] in skeletal muscle. No other statistically significant bivariate correlations were consistently found. Females had a greater relative PFO compared to males: 7.1±1.9 vs 4.5±1.3 and 7.3±1.7 vs 4.8±1.2 mgkg FFM-1min-1)] in the adipose tissue (n=14) and skeletal muscle (n=12) sub-groups, respectively (p<0.05). No statistically significant sex differences were found in the content of these proteins. The regulation of PFO may involve processes relating to intramyocellular triglyceride hydrolysis and mitochondrial fatty acid transport, and adipose tissue is likely to play a more minor role than muscle. Sex differences in fat metabolism are likely to be due to factors other than the resting content of proteins in skeletal muscle and adipose tissue relating to triglyceride hydrolysis and fatty acid transport.