〔Abstract〕 To establish and identify hepatocyte-specific transmembrane protein 121 (Tmem121) knockout mice. Methods The hepatocyte-specific Tmem121 knockout mice (Tmem121flox/flox/Cre, Tmem121ΔHep) were obtained by crossbreeding of Tmem121flox/+/Cre and Tmem121flox/flox mice, which were generated using the CRISPR/Cas9 and Cre/Loxp systems. The genotype was verified by PCR using genomic DNA extracted from mouse tails as template. The growth, reproduction and organ development of both control and knockout mice were observed and analyzed. PCR and Western blot methods were performed to assess the knockout efficiency of Tmem121 in mouse primary hepatocytes. CellMask™ Deep Red plasma membrane staining was employed to compare the morphological differences in primary hepatocytes between control and knockout mice. Results Tmem121flox/flox/Cre mice were successfully obtained according to genotype identification analysis, and there were no significant differences between control and knockout mice in body mass, reproductive ability, growth and development of liver. The specific knockout of Tmem121 gene in primary hepatocytes did not significantly affect the morphological structure or pathological characteristics of liver tissue. However, compared to the control group, the levels of Tmem121 mRNA and protein in the primary hepatocytes of the knockout group were significantly reduced (P<0.01). CellMask™ Deep Red plasma membrane staining indicated that the proportion of binucleated hepatocytes in Tmem121-deficient mice significantly increased (P<0.05), while the cell area was significantly reduced (P<0.001). Conclusion Hepatocyte-specific Tmem121 knockout mice are successfully constructed, which provides an animal model for further exploration of the function and mechanism of Tmem121 gene in liver diseases.