Mechanomedicine represents a potential biocompatible method in cancer therapy. In particular, the use of low-frequency mechanical vibration previously proved to trigger apoptosis of the human epidermoid carcinoma A431 cell line. In this study, we further characterized the metabolic and oxidative stress responses triggered by 1 h of 20 Hz mechanical vibration stimulus to A431 prior to cell death. Our results indicate that cell death may be related to the decrease of glucose consumption rate and the higher expression of reactive oxygen species right after mechanical stimulation (0 h). The overexpression of HMGB1 and HSP70 coding genes signified the increase of A431 cell stress. However, HMGB1 and HSP70 expression decreased at 24 h after mechanical vibration, along with the progression of cell death. We also observed cell morphology changes on A431 cells following vibration which might be indicative of A431 death by apoptosis. The emergence of these stress responses suggests that several pathways are connected to promote cancer cell death. The discovery of A431 cellular stress symptoms which lead to apoptotic death may clarify the usefulness of mechanical vibration in cancer treatment as a novel application of biomechanical manipulation.