Morphology together with the capability to respond to surrounding stimuli are the key elements governing the spatial interaction of a living cell with the environment. In this respect, biomechanical stimulation can trigger significant physiological cascades that can potentially modulate or interact with toxicity. Deoxynivalenol (DON, vomitoxin) is one of the most prevalent mycotoxins produced by Fusarium spp. and it was used to explore the delicate interaction between biomechanical stimulation and cytotoxic processes in A431 cells. In fact, in addition of being a well-known food contaminant, DON is becoming a relevant toxin also for other organ systems. The combination between biomechanical stimulation and the mycotoxin revealed how DON can impair crucial functions like cellular morphology and lysosome trafficking at concentrations far below those known to be cytotoxic in routine toxicity studies. Moreover, sub-toxic concentrations of DON (0.1-1 μM) impaired the capability of A431 cells to respond to a biomechanical stimulation that normally sustains trophic effects in this cell line. Ultimately, the effects of DON (0.1-10 μM) were partially modulated by the application of uniaxial stretching (0.5Hz, 24h, 15% deformation) suggesting a deep cross-talk between the cytotoxic potential of the mycotoxin and the biomechanical stimulation.