Cancer cells encounter various mechanical stresses from the extracellular microenvironment. Understanding how cancer cells adapt to these stresses is critical for developing innovative anticancer therapies. In this study, we designed cell culture systems that imposed five types of mechanical stresses-fluid viscosity, fluid shear, matrix adhesion, stretching, and confinement pressure, and studied the proliferation and migration responses of 8 cell lines representing 6 cancer types under these conditions. We also performed proteomic analysis of the 8 cancer cell lines under different mechanical force conditions. Among them, we identified 10,790 proteins in the fluid viscosity and matrix adhesion stress tests, of which 2,931 showed significant mechanical force responses. These included 1,413 proteins that changed under high fluid viscosity stress, 1,264 proteins that changed under high matrix adhesion stress, and 286 proteins that changed in both conditions. Correlation analysis showed that there were different mechanical response pathways between glioma and liver cancer cells compared with lung cancer, breast cancer, and colorectal cancer. In addition, differential protein expression was not only related to cell migration, but also to mitochondrial inner membrane function, matrix ATP binding, and energy metabolism, highlighting the important role of mitochondria in managing cellular processes under stress. Notably, ITIH4 and A2MG were upregulated in all cells under fluid viscosity stress, with the increase in ITIH4 further validated by WB, indicating its role as a mechanosensitive factor. In addition, FN1 protein was significantly upregulated in cancer cells facing high matrix adhesion stress, indicating its involvement in responding to this specific stress. The comprehensive approach of this study combining mechanical stress systems with proteomics facilitated the discovery of novel mechanoresponsive molecules. Future studies will aim to identify key proteins in stress response pathways, explore their molecular functions, and elucidate the mechanosensitivity mechanisms of cancer cells, thereby facilitating the development of targeted anticancer therapies.