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We previously demonstrated a close relevance between a single nucleotide polymorphism, rs290487 of TCF7L2 gene, in donor grafts and new-onset diabetes mellitus in liver transplant recipients. In this study, we aim to make a functional analysis of hepatic rs290487 variant in glucose metabolism homeostasis.We used CRISPR/Cas 9 technology to construct a variant type in rs290487 locus in PLC-PRF-5 cell line. The base Cytosine in rs290487 SNP site of TCF7L2 gene was replaced with Thymine in PLC-PRF-5 cell line. A transcriptome sequencing analysis was preformed to detect the differential expression profiles between C/C and T/T genotype PLC-PRF-5 cells.Compared to C/C genotype, T/T genotype PLC-PRF-5 cells showed increased glucose uptake and insulin sensitivity, and decreased glucose production (all P < 0.05). There was a significant decrease of TCF7L2 mRNA expression and protein content after C to T transition at rs290487 in PLC-PRF-5 cells (fold change = 0.61, adjusted P < 0.001). In alternative splicing analysis, C/C genotype lost expression of variant 5 and showed significantly decreased expression of variant 2 as compared to T/T genotype (both P < 0.05). mRNA-seq revealed 896 and 714 genes significantly up- and down-regulated, respectively, in T/T genotype compared with C/C according to a criteria that adjusted P value < 0.05 and fold change > 2. KEGG analysis demonstrated a very strong enrichment in metabolic pathways with 553 significantly differentially expressed genes involved. We focused on glucose metabolism and found significant up- (e.g., LEPR, GPD1, G6PC, and GYS2) and down-regulated key genes (e.g., RBP4, PDK4, HK2, and FOXA2) after C to T transition. Of note, 30 genes in drug metabolism-cytochrome P450 pathway and nuclear factor of activated T (NFAT) family (key calcineurin targets) were significantly down-regulated after C to T transition. This may affect the calcineurin inhibitors metabolism and be associated with new-onset diabetes mellitus in liver transplant recipients.TCF7L2 gene rs290487 is a functional intronic variant and modulates hepatic glucose metabolism.