Doxorubicin is a broad-spectrum chemotherapy drug commonly used in clinical practice. Improving the delivery efficiency of doxorubicin can help improve the therapeutic effect of tumor and reduce the corresponding side effects. The delivery of doxorubicin based on nano-drug loading technology can provide important help for targeted killing of tumor cells. Tumor development is usually accompanied by the changes in the stiffness of extracellular matrix. The effect of substrate stiffness on the endocytic behavior of doxorubicin based on drug-loading nanoparticle technology was explored by the cellular activity regulated by doxorubicin endocytosis and the changes in the expression of important proteins associated with endocytosis. The substrate stiffness can be regulated by adjusting the ratio of PDMS binder and curing agent. The results of the experiment showed that the cell activity on the substrate with appropriate stiffness (Youngs modulus of 0.578, 0.815 and 1.986 MPa) was inhibited, indicating that the transport of doxorubicin was more efficient in this range of substrate stiffness. To explore the underlying mechanism of substrate stiffness regulation of doxorubicin endocytosis, we explored the expression of αVβ5 and NUMB, which are closely related to the endocytosis process. From the results of immunofluorescence protein and Western blot experiments, the fluorescence intensity of the αVβ5 protein responsible for anchoring clathrin on the substrate with a Youngs modulus of 1.986 MPa was the highest; Within the appropriate stiffness range, the fluorescence intensity of NUMB protein was significantly increased, indicating that increased expression of NUMB and αVβ5 regulated by substrate stiffness mediates doxorubicin transport, thereby affecting cell viability.This work provides a reference for further improving the efficacy of chemotherapy drugs in the treatment of tumors.