The carnitine shuttle system (CSS) plays a crucial role in the transportation of fatty acyls during fatty acid β-oxidation for energy supplementation, especially in cases of high energy demand, such as in cancer. In this study, to systematically characterize alterations of the CSS in hepatocellular carcinoma (HCC), acylcarnitine metabolic profiling was carried out on 80 pairs of HCC tissues and adjacent noncancerous tissues (ANTs) by using ultra-performance liquid chromatography coupled to mass spectrometry. Twenty-four acylcarnitines classified into five categories were identified and characterized between HCCs and ANTs. Notably, increased saturated long-chain acylcarnitines (LCACs) and decreased short- and medium-chain acylcarnitines (S/MCACs) were simultaneously observed in HCC samples. Subsequent correlation network and heatmap analysis indicated low correlations between LCACs and S/MCACs. The mRNA and protein expressions of carnitine palmitoyltransferase 2 (CPT2) was significantly downregulated in HCC samples, whereas CPT1A expression was not significantly changed. Correspondingly, the relative levels of S/MCACs were reduced and those of LCACs were increased in BEL-7402/CPT2-knockdown cells compared to negative controls. Both results suggested that decreased shuttling efficiency in HCC might be associated with downregulation of CPT2. In addition, decreases in the mRNA expression of acetyl-CoA acyltransferase 2 were also observed in HCC tissues and BEL-7402/CPT2-knockdown cells, suggesting potential low β-oxidation efficiency, which was consistent with the increased expression of stearoyl-CoA desaturase 1 in both samples. The systematic strategy applied in our study illustrated decreased shuttling efficiency of the carnitine shuttle system in HCC and can provide biologists with an in-depth understanding of β-oxidation in HCC.