Objective To investigate the detailed molecular mechanism of matrix stiffness regulating cell drug resistance. Methods Polyacrylamide hydrogels of soft substrate (10 kPa), hard substrate (38 kPa) and rigid substrate (57 kPa) with different matrix stiffness were configured to simulate the physical matrix stiffness at different stages of breast cancer in vivo. Results The cell proliferation rate of the hard substrate was significantly higher than that of the soft and rigid substrates. The intracellular endocytosis was significantly lower on the hard substrate. The YAP nucleus translocation increased significantly on the hard substrate, compared with the soft and the rigid substrates, indicating that YAP was a key molecule involved in drug resistance of tumor cells. Conclusions Matrix stiffness could regulate the drug resistance of breast cancer cells through YAP activation. This study not only provides a new direction for elucidating the mechanism of drug resistance, but also lays a new foundation for the drug delivery system of breast cancer treatment.