Abstract: The aim of the present study was to obtain a stable, spontaneous insulin-resistant adipocyte model by altering the gene sequence of glucose transporter 4 (GluT4) which resulted in the dysfunction of glucose transmembrane transport in 3T3-L1 cell line. In this study, the GluT4 coding region of 3T3-L1 was directionally cleaved by CRISPR/Cas9 protein, and the stably mutated cell lines were screened by flow cytometry and gene sequencing. The proliferation activities of the selected cell lines were detected by CCK-8 method. Subsequently, the cells were treated with adipogenic differentiation reagent and identified by oil red O staining. The glucose uptake of cells was detected under basal and insulin-stimulated state to determine the insulin resistant level. qPCR and Western blotting were used to detect the expression of essential genes in the process of adipogenic differentiation of insulin-resistant cells and control cells. The results showed that the 13 bp deletion in the 9th exon resulted in dysfunction of GluT4 protein in 3T3-L1 cells without affecting cell proliferation. Sever and stable spontaneous insulin resistance and impaired glucose uptake occurred as we expected. Gene transcription associated with adipogenic differentiation and lipid synthesis were significantly downregulated (P<0.05 or P<0.01) compared with controls. In summary, GluT4 dysfunction hinders insulin-stimulated glucose uptake, resulting in a spontaneous insulin resistance in adipocytes. This study verified the stability and effectiveness of the insulin resistance adipocyte model from multiple perspectives, provided a new material for studies related to adipose metabolism and insulin resistance.