Aims: To investigate the effects of ABCB1 DNA methylation in donors on individual differences in tacrolimus blood concentrations following liver transplantation. Methods: Twenty-three donor liver samples carrying the CYP3A5*3/*3 genotype were classified into two groups based on the initial tacrolimus concentration/dose (C0/D) ratio following liver transplantation. ABCB1 mRNA levels in liver tissues and HepG2 cells were determined by qRT-PCR. DNA methylation status in liver tissues and HepG2 cells was determined using Illumina 850 methylation chip sequencing technology and pyrosequencing. 5-Aza-2dC was used to reverse methylation in HepG2 cells. Intracellular tacrolimus concentrations were determined by liquid mass spectrometry. Results: Genome-wide methylation sequencing and pyrosequencing analyses showed that the methylation levels of three ABCB1 CpG sites (cg12501229, cg00634941, and cg05496710) were significantly different between groups with different tacrolimus C0/D ratios. ABCB1 mRNA expression in donor livers was found to be positively correlated with tacrolimus C0/D ratio (r = 0.458, P < 0.05). After treatment with 5-Aza-2-Dc, the methylation levels of the ABCB1 CpG sites in HepG2 cells significantly decreased, and this was confirmed by pyrosequencing; there was also a significant increase in ABCB1 transcription, and this most likely induced a decrease in intracellular tacrolimus concentrations. Conclusions: ABCB1 CpG site methylation affects tacrolimus metabolism in humans by regulating ABCB1 expression. Therefore, ABCB1 DNA methylation in donor livers might be an important epigenetic factor that affects tacrolimus blood concentrations following liver transplantation.