The unique dietary fiber composition with high contents of β-glucan contributes to the health-promoting properties of oat and barley and may mediate a reduction of colon cancer risk. In the present study, chemopreventive effects of oat and barley (beta®barley) kernels were investigated. In order to address the impact of thermal processing on these effects, kernels were roasted (150-180 °C, approx. 20 min), digested and fermented using an In Vitro human digestion model. Concentrations of short-chain fatty acids (SCFA) and ammonia were determined in fermentation supernatants (FS). Growth inhibition, apoptosis, DNA integrity and gene expression of catalase were analyzed in LT97 colon adenoma cells. Concentrations of SCFA, particularly butyrate, were higher in oat/barley FS (2.2-fold, on average), while ammonia levels were significantly lower (0.7-fold, on average) than in the fermentation control. Treatment of LT97 cells with FS of oat/barley kernels led to a significant time- and dose-dependent growth reduction, a significant increase in caspase-3 activity and enhanced levels of catalase mRNA, without exhibiting genotoxic effects. In general, the results indicate a chemopreventive potential of In Vitro fermented oat and waxy winter barley mediated mainly by growth inhibitory and apoptotic effects, which are preserved after thermal processing.