Although carnitine palmitoyltransferase-I (CPT-I) is generally regarded to present a major rate-controlling site in mitochondrial β-oxidation, it is incompletely understood whether CPT-I is rate-limiting in the overall long-chain fatty acid (LCFA) flux in the heart. Another important site of regulation of the LCFA flux in the heart is trans-sarcolemmal LCFA transport facilitated by CD36 and FABPpm. Therefore, we explored to what extent a chronic pharmacological blockade of the LCFA flux at the level of mitochondrial entry of LCFA-CoA would affect sarcolemmal LCFA uptake. Rats were injected daily with saline or etomoxir, a specific CPT-I inhibitor, for 8 days at 20 mg/kg body mass. Etomoxir-treated rats displayed a 44% reduced cardiac CPT-I activity. Sarcolemmal contents of CD36 and FABPpm, as well as the LCFA transport capacity were not altered in hearts of etomoxir-treated versus control rats. Furthermore, rates of LCFA uptake and -oxidation and glucose uptake by cardiac myocytes from etomoxir-treated rats were not different from control rats, neither under basal nor under acutely induced maximal metabolic demands. Finally, hearts from etomoxir-treated rats did not display triacylglycerol accumulation. Therefore, CPT-I appears not to present a major rate-controlling site in total cardiac LCFA flux. Likely, sarcolemmal LCFA entry rather than mitochondrial LCFA-CoA entry is a promising target for normalizing LCFA flux in cardiac metabolic diseases.