Penicillium roqueforti produces mycotoxins including PR toxin, which is a food and feed contaminant. In this study, PR toxin was purified from culture material of the Penicillium roqueforti F43-1 strain. Toxic effects were evaluated in undifferentiated human Caco-2 intestinal epithelial cells and THP-1 monocytic immune cells. To understand the mechanisms involved in PR-toxin toxicity, cell death and pro-inflammatory gene expression were studied. In addition, PR toxin degradation was assessed. Cytotoxicity studies showed a dose-dependent effect of PR toxin and the calculated mean cytotoxic concentration (IC50) concentrations were for Caco-2 and THP-1 cells >12.5 and 0.83 μM, respectively. Gene expression studies showed that tumour necrosis factor-α expression was significantly increased after 24 h exposure to 312 μM PR toxin. PR toxin induced necrosis on THP-1 cells after 3 h exposure. In the cell culture system, the PR toxin showed a 10-fold reduction in PR toxin concentration within 48 h, indicating that PR toxin was degraded by THP-1. To conclude, PR toxin appears to be one of the most cytotoxic P. roqueforti mycotoxins on Caco-2 and/or THP-1 cells and induces in THP-1 cells both necrosis and an inflammatory response.