Clostridium difficile toxin A (TcdA) impairs the intestinal epithelial barrier, increasing the mucosa permeability and triggering a robust inflammatory response. Lathyrus sativus diamino oxidase (LSAO) is a nutraceutical compound successfully used in various gastrointestinal dysfunctions. Here, we evaluated the LSAO (0.004–0.4 μM) ability to counter TcdA‐induced (30 ng/mL) toxicity and damage in Caco‐2 cells, investigating its possible mechanism of action. LSAO has improved the transepithelial electrical resistance (TEER) score and increased cell viability in TcdA‐treated cells, significantly rescuing the protein expression of Ras homolog family members, A‐GTPase (RhoA‐GTPase), occludin, and zonula occludens‐1 (ZO‐1). LSAO has also exhibited an anti‐apoptotic effect by inhibiting the TcdA‐induced expression of Bcl‐2‐associated X protein (Bax), p50 nuclear factor‐kappa‐B (p50), p65nuclear factor‐kappa‐B (p65), and hypoxia‐inducible transcription factor‐1 alpha (HIF‐1α), and the release of tumor necrosis factor‐alpha (TNF‐α), interleukin‐6 (IL‐6), and vascular endothelial growth factor (VEGF) in the cell milieu. Our data showed that LSAO exerts a protective effect on TcdA‐induced toxicity in Caco‐2 cells, placing itself as an interesting nutraceutical to supplement the current treatment of the Clostridium difficile infections.