OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD) has become a common liver disorder caused by lipid accumulation and insulin resistance (IR). Acylcarnitines have become a new biomarker of IR. However, their roles in NAFLD are still poorly studied. Thus, we performed a targeted metabolomic analysis to study the level of plasma acylcarnitines in patients with NAFLD. PATIENTS AND METHODS: The levels of 34 plasma acylcarnitines were measured by a targeted metabolomic approach in NAFLD patients (n = 50) and in healthy control subjects (n = 50) by liquid chromatography-tandem mass spectrometry. Detailed demographic and clinical characteristics of all subjects were also analyzed. RESULTS: The clinical presentation of IR was identified in the NAFLD group but not in the healthy control group. Significant differences were found in the levels of several short-, medium- and long-chain acylcarnitines. A high degree of correlation (r> 0.7) was found between even-numbered-carbon long-chain acylcarnitines in NAFLD patients. The area under the receiver operator characteristic of long-chain acylcarnitines, especially C20 (AUC= 0.952), C16:1 (AUC= 0.949) and C14:1OH (AUC=0.944) acylcarnitines, was greater in NAFLD patients than in healthy control subjects. CONCLUSIONS: The accumulation and disorders of acylcarnitines are associated with NAFLD. A positive correlation between even-numbered-carbon long-chain acylcarnitines was found, and these even-numbered-carbon longchain acylcarnitines. could be used as potential novel screening markers for nonalcoholic fatty liver disease.