〔Abstract〕 Objective To explore the role of m6A demethylase FTO in the resistance of breast cancer cell to trastuzumab and the effect of the new anti-HER2 antibody HuA21 on the proliferation of drug-resistant breast cancer cell. Methods Trastuzumab-resistant cell line BT474/TR was prepared. The morphology of BT474/TR and BT474 cells were observed under optical microscope. MTT, immunofluorescence and flow cytometry were used to detect cell proliferation, the expression of proliferation-related antigen Ki67 and cell cycle. qPCR and Western blot methods were used to determine the expression of m6A-related enzymes FTO and METTL3. Results BT474/TR showed pyknosis of the nucleus, the pseudopodia disappeared, and the cell proliferation rate decreased. As BT474 transited to BT474/TR with trastuzumab treatment, Ki67 expression and S phase cell proportion gradually decreased(P<0.05), and FTO expression gradually increased(P<0.05). However, there was no difference in METTL3 expression. The FTO inhibitor MA2 promoted the BT474/TR proliferation and increased its sensitivity to trastuzumab treatment(P<0.05). HuA21 antibody had an inhibitory effect on the proliferation of BT474/TR cells(P<0.05), and the combination of MA2 and trastuzumab had a better inhibitory effect on BT474/TR cells(P<0.05). Conclusion There is a correlation between the resistance of HER2-positive breast cancer cell to trastuzumab and FTO gene expression and the inhibition of FTO activity can enhance the sensitivity of drug-resistant cells to trastuzumab treatment. Moreover, HuA21 antibody has an inhibitory effect on the proliferation of BT474/TR cells.