Phenotypic plasticity is posed to be a vital trait of cancer cells such as circulating tumor cells, allowing them to undergo reversible or irreversible switching between phenotypic states important for tumorigenesis and metastasis. While irreversible phenotypic switching can be detected by studying the genome, reversible phenotypic switching is often difficult to examine due to its dynamic nature and the lack of knowledge about its contributing factors. In this study, we demonstrate that culturing cells in different physical environments, stiff, soft, or suspension, induced a phenotypic switch in prostate cancer cells via mechanotransduction. The mechanosensitive phenotypic switching in prostate cancer cells was sustainable yet reversible even after long-term culture, demonstrating the impact of mechanical signals on prostate cancer cell phenotypes. Importantly, such a mechanotransduction-mediated phenotypic switch in prostate cancer cells was accompanied by decreased sensitivity of the cells to paclitaxel, suggesting a role of mechanotransduction in the evolution of drug resistance. Multiple signaling pathways such as p38MAPK, ERK, and Wnt were found to be involved in the mechanotransduction-induced phenotypic switching of prostate cancer cells. Given that cancer cells experience different physical environments during disease progression, this study provides useful information about the important role of mechanotransduction in cancer, and how circulating tumor cells may be capable of continuously changing their phenotypes throughout the disease process.