The mycotoxin citrinin (CTN) is a natural contaminant of various human foods that may produce serious adverse health problems. Several studies demonstrated that citrinin exerts cytotoxic and genotoxic effects in both in vivo and in vitro systems. However, the precise mechanisms of action (MOA), particularly in intestinal cells remain unclear. The aim of the present study was to examine the precise MOA of citrinin in vitro. Data demonstrated that CTN significantly decreased the number of viable human intestinal HCT116 cells and induced apoptotic events including (1) decrease in ΔѰm indicative of mitochondrial membrane permeabilization, (2) activation of caspase 3, (3) elevated production of reactive oxygen species (ROS) and (4) relative persistence of plasma membrane integrity. Further, the genetic deficiency of the pro-apoptotic protein Bax protected cells against CTN-induced apoptosis, indicating that Bax is required for CTN-mediated toxicity. It was also found that CTN triggered endoplasmic reticulum (ER) stress and activated different arms of the unfolded protein response (UPR) as demonstrated by increase in expression of GRP78 (glucose-regulated protein-78), GRP94 (glucose-regulated protein-94), GADD34 (growth arrest and DNA damage-inducible protein-34), the protein disulfide isomerase associated 6 (PDIA6), CHOP (C/EBP-homologous protein) and the splicing of XBP1 (X-Box Binding Protein 1). Pretreatment of cells with the chemical chaperone 4-phenylbutyrate (PBA), known to alleviate ER stress, prevented significantly the apoptotic process triggered by CTN. Taken together, these results suggest that CTN exerts its cytotoxic effects in HCT116 cells by inducing apoptosis, at least in part, through induction of ER stress.