The objective of this study was to determine the effects of NEFA and glucose on carnitine palmitoyltransferase-I (CPT-I) mRNA expression in cultured bovine hepatocytes using real-time reverse transcription polymerase chain reaction and ELISA methods. The results indicated that CPT-I transcription increased gradually, but that CPT-I translation was not significantly changed, with glucose concentrations ranging from 0 to 3.0 mmol/L (P<0.01). Furthermore CPT-I transcription and translation were enhanced significantly when the NEFA concentrations increased from 0 to 1.2 mmol/L and decreased significantly when the NEFA concentrations increased from 1.2 to 4.8 mmol/L (P<0.01). A high concentration NEFA was found to reduce fatty acid oxidation, potentially explaining the development from NEB to ketosis in dairy cows.