Ectopic deposition of fat in skeletal muscle is a feature of metabolic syndrome, but its specific association with very-low-density lipoprotein (VLDL)-apolipoprotein (apo) B-100 metabolism remains unclear. We examined the association between skeletal muscle fat content and VLDL-apoB-100 kinetics in 25 obese subjects, and the responses of these variables to weight loss. The fat contents of liver, abdomen and skeletal muscle were determined by magnetic resonance imaging, and VLDL-apoB-100 kinetics were assessed using stable isotope tracers. In obese subjects who were insulin sensitive (homeostasis model assessment, HOMA, score ≤ 2.6, n = 12), skeletal muscle fat content was significantly associated with hepatic fat content (r = 0.636), energy intake (r = 0.694), plasma triglyceride (r = 0.644), apoB-100 (r = 0.529), glucose (r = 0.622), VLDL-apoB-100 concentrations (r = 0.860), VLDL-apoB-100 fractional catabolic rate (FCR; r = -0.581) and VLDL-apoB-100 secretion rate (r = 0.607). These associations were not found in obese subjects who were insulin resistant (HOMA score >2.6, n = 13). Of these 25 subjects, 10 obese subjects underwent a 16-week weight loss program. The low-fat diet achieved significant reduction (p < 0.05) in body weight, visceral and subcutaneous fat areas, liver and skeletal muscle fat, energy intake, triglyceride, insulin, HOMA score, VLDL-apoB100 concentrations and VLDL-apoB100 secretion rate. The percentage reduction of skeletal muscle fat with weight loss was significantly associated with the corresponding changes in VLDL-apoB100 concentration (r = 0.770, p = 0.009) and VLDL-apoB-100 secretion (r = 0.682, p = 0.030). Skeletal muscle fat content is associated with VLDL-apoB-100 transport. Weight loss lowers skeletal muscle fat and VLDL-apoB-100 secretion.