Our previous study has demonstrated that procyanidin A₁ (A₁) and its simulated digestive product (D-A₁) can prevent acrylamide (ACR)-induced cytotoxicity in small intestine cells. However, the potential mechanism remains poorly understood. In this study, ACR treatment was found to increase the levels of 8-hydroxy-deoxyguanine (8-OHdG) and phosphorated histone H₂AX (γH₂AX), two DNA damage markers, thereby resulting in cell cycle arrest in the G2/M phase; whereas both A₁ and D-A₁ could prevent the phosphorylation of ataxia telangiectasia mutated (ATM) and checkpoint kinase 2 (Chk2), and then regulate the expression of G2/M phase-related proteins, finally maintaining normal cell cycle progression. Moreover, A₁ and D-A₁ could increase the B cell lymphoma 2 (Bcl-2)/Bcl2-associated X (Bax) ratio and decrease the expression of cleaved caspase-3 and cleaved caspase-9 proteins to alleviate ACR-induced cell apoptosis, which might be related to the inhibition of the mitogen-activated protein kinase (MAPK) pathway. More importantly, A₁ showed no remarkable variation in inhibitory effect before and after digestion, indicating that it can endure gastrointestinal digestion and may be a promising phytochemical to alleviate ACR-induced intestinal cell damage.