The effects of the black rice anthocyanidin monomer cyanidin-3-O-glucoside (C3G) and its lauric acid enzymatic acylation product cyanidin-3-(6″-lauroyl) glucose (C3G-C12) on cholesterol absorption and transport in Caco-2 cells and its possible mechanism of action were investigated. In this study, C3G was extracted from black rice anthocyanidins using semi-preparative–high performance liquid chromatography (semi-HPLC), and the monomer was identified using liquid chromatography–mass spectrometry (LC-MS/MS). C3G was used as raw material and enzymatically acylated with lauric acid and Novozymes 435. The acylated products were further separated via semi-HPLC, and the monomer was verified via LC-MS/MS and Fourier transform infrared spectroscopy. A 21-day Caco-2 intestinal epithelial cell model was established. The cells were incubated with 50, 100, and 150 μg•mL-1 C3G and C3G-C12 and 20 μmol•L-1 cholesterol absorption inhibitor ezetimibe to perform transport experiments. HPLC was used to detect the cellular cholesterol absorption, and fluorescence quantitative polymerase chain reaction was used to detect NPC1L1 mRNA expression. The results showed that both inhibits cholesterol transport of Caco-2 cells in a concentration-dependent manner. Specifically, 150 μg•mL-1 C3G and C3G-C12 reduced cholesterol absorption by 46.21% and 56.36%, respectively, and NPC1L1 mRNA expression by 56.93% and 59.76%, respectively. In summary, C3G-C12 more effectively inhibited cholesterol absorption than C3G, which might be related to its inhibition of NPC1L1 mRNA expression. This study provides a theoretical basis for the enzymatic acylation modification of black rice anthocyanins and their role in regulating intestinal cholesterol metabolism.