The role of heart-type cytosolic fatty acid binding protein (H-FABP) in mediating whole-body and muscle specific long chain fatty acid (LCFA) and glucose utilization was examined using exercise as a phenotyping tool. Catheters were chronically implanted in a carotid artery and jugular vein of wild type (WT, n=8), heterozygous (H-FABP^+/-, n=8) and null (H-FABP^-/-, n=7) chow fed, C57BL/6J mice and allowed to recover for 7d. Following a 5h fast, conscious, unrestrained mice were studied during 30min of treadmill exercise (0.6mi/h). A bolus of [¹²⁵I]- 15-(-iodophenyl)-3-R,S-methylpentadecanoic acid and 2-deoxy[³H]glucose was administered to obtain rates of whole body metabolic clearance (MCR) and indices of muscle LCFA (R_f) and glucose (R_g) utilization. Fasting, non-esterified fatty acids (mM) were elevated in H-FABP^-/- (2.2±0.9 vs. 1.3±0.1 and 1.3±0.2 for WT and H-FABP^+/-). During exercise, blood glucose increased in WT (11.7±0.8) and H-FABP^+/- (12.6±0.9) while H-FABP^-/- developed overt hypoglycemia (4.8±0.8). Examination of tissue specific and whole body glucose and LCFA utilization demonstrated a dependency on H-FABP with exercise in all tissues examined. Reductions in H-FABP led to decreasing exercise-stimulated R_f and increasing R_g with the most pronounced effects in heart and soleus muscle. Similar results were seen for MCR with decreasing LCFA and increasing glucose clearance with declining levels of H-FABP. These results show that in vivo, H-FABP has reciprocal effects on glucose and LCFA utilization and whole body fuel homeostasis when metabolic demands are elevated by exercise.