Intestinal cells are able to continuously integrate response to multiple stimuli/stressors; these include the concomitant activation of “chemically driven” pathways, of paramount importance in the response to toxicants, as well as physical stimulation derived from motility. Altertoxin II (ATXII, 0.1, 1 and 10 µM), a mycotoxin produced by the food contaminant fungus Alternaria alternata was studied in HT-29 intestinal adenocarcinoma cells and in non-transformed intestinal epithelial cells, HCEC. One-hour incubation with ATXII was sufficient to trigger irreversible cytotoxicity in both cell types, as well as to modify cellular responses to concomitant pro-oxidant challenge (H2O2, 100–500 µM, DCF-DA assay) suggesting that even relatively short-time exposure of the intestinal cells could be sufficient to alter their functionality. Combination of ATXII (1 µM) with physical stimulation typical of the intestinal compartment (shear stress) revealed differential response of tumor-derived epithelial cells HT-29 in comparison to HCEC, in particular in the localization of the transcription factor Nrf2 (NF-E2-related factor 2). Moreover, ATXII reduced the migratory potential of HCEC as well as their membrane fluidity, but had no respective impact on HT-29 cells. Taken together, ATXII appeared to alter predominantly membrane functionality in HCEC thus hampering crucial functions for cellular motility/turnover, as well as barrier function of healthy intestinal cells and had very limited activity on the tumor counterparts.