The possibility to combine untargeted proteomic workflows to more classical experimental approaches is continuously opening new insights in all branches of biological sciences. Deoxynivalenol (vomitoxin, DON) is a secondary metabolite produced by Fusarium spp. fungi and it is one of the most recurrent mycotoxins worldwide. DON is known to inhibit protein synthesis and as such to interact with the different cell types in multiple and complex ways. For the purpose of this study epidermoid squamous cell carcinoma cells A431 and primary human HEKn cells were incubated with DON for 24h and toxin dependent alteration of the proteome profile was observed in the nuclear and cytoplasmic fraction. In A431 cells, DON significantly down-regulated ribosomal proteins, as well as mitochondrial respiratory chain elements (OXPHOS regulation) and transport proteins (TOMM22; TOMM40; TOMM70A). In line with the impairment of the mitochondrial function, altered metabolic capability was observed, with particular target of the lipid synthesis machinery. Effect of the mycotoxin on cell membrane was verified by confocal microscopy (morphology) and by membrane fluidity measures (biophysical properties). The toxicological relevance of these findings was independently confirmed with the primary human keratinocytes HEKn.