Objective The insertion mutation of human epidermal growth factor receptor 2 (ERBB2) (p. Pro780_Tyr781insGSP)leads to the emergence of rapatinib resistance through activating protein kinase B (Akt) signaling pathway, in order to explore the potential mechanism of the mutation. Methods Polymerase chain reaction (PCR), restriction enzyme and ligation were used to construct control group pHelper. CopGFP, wild group pHelper. GV358-ERBB2, mutant group pHelper. GV492-ERBB2 plasmid to transfect 293T cells, collect supernatant and package lentivirus. The titer was 108, transfected in MCF-7 cells with a fusion rate of 70%-80%, and the drug was sieved in 1 mg/L puromycin to construct a stable strain, by fluorescence microscopy and western blotting to detect transfection efficiency. The experiment was divided into three groups: ERBB2-NC group, ERBB2-WT group and ERBB2-MT group. The half maximal inhibitory concentration (IC50) of MCF-7 cells treated with lapatinib for 48 h was detected by cell proliferation cell counting kit-8 (CCK-8) method. The expression of ERBB2 related signaling pathway protein was detected by Western blot after lapatinib treatment. The effect of lapatinib on cell cycle and apoptosis was determined. Results The vector was successfully constructed and sequenced correctly. The wild type ERBB2 (ERBB2-WT) and mutant ERBB2 (ERBB2-MT) stable transformants were successfully constructed. The cell proliferation inhibition experiment showed that the IC50 in the ERBB2-MT group was (90.41±7.47) μmol/L, which was significantly higher than that of the ERBB2-WT group (43.94±9.84) μmol/L. Compared with the other two groups, the ERBB2-MT group showed significant resistance to lapatinib (F=55.530, P<0.01). Cell growth was promoted by increasing the phosphorylation level of Akt, The ERBB2-MT group (326.00±36.16) cell clones increased compared with the ERBB2-WT group (201.00±10.03) (t=7.478, P<0.05). And advanced Apoptosis (WT: 22.29±1.53, MT: 10.89±2.42, t=6.905, P<0.01) and enhanced cell proliferation (WT: 18.95±0.73, MT: 33.05±2.69, t=8.774, P<0.01)developed resistance to lapatinib. Conclusion The results indicate that the insertion mutation of ERBB2-MT is a cancer-promoting mutation that is resistant to the treatment of lapatinib by activating phosphorylation of the Akt signaling pathway. Key words: Breast cancer; Insertion mutation; Resistance