Deoxynivalenol (DON) is one of the most prevalent mycotoxin contaminants, which posing a serious health threat to animals and humans. Previous studies have found that individually supplemented probiotic or glycyrrhinic acid (GA) could degrade DON and alleviate DON-induced cytotoxicity. The present study investigated the effect of combining GA withSaccharomyces cerevisiae(S. cerevisiae) andEnterococcus faecalis(E. faecalis) using orthogonal design on alleviating IPEC-J2 cell damage induced by DON. The results showed that the optimal counts ofS. cerevisiaeandE. faecalissignificantly promoted cell viability. The optimal combination for increasing cell viability was 400 µg/mL GA, 1 × 106CFU/mLS. cerevisiaeand 1 × 106CFU/mLE. faecalisto make GAP, which not only significantly alleviated the DON toxicity but also achieved the highest degradation rate of DON (34.7%). Moreover, DON exposure significantly increased IL-8, Caspase3 and NF-κB contents, and upregulated the mRNA expressions of Bax, Caspase 3, NF-κB and the protein expressions of Bax, TNF-α and COX-2. However, GAP addition significantly reduced aforementioned genes and proteins. Furthermore, GAP addition significantly increased the mRNA expressions of Claudin-1, Occludin, GLUT2 and ASCT2, and the protein expressions of ZO-1, Claudin-1 and PePT1. It was inferred that the combination of GA,S. cerevisiae, andE. faecalishad the synergistic effect on enhancing cell viability and DON degradation, which could protect cells from DON-induced damage by reducing DON cytotoxicity, alleviating cell apoptosis and inflammation via inhibiting NF-κB signaling pathway, improving intestinal barrier function, and regulating nutrient absorption and transport. These findings suggest that GAP may have potential as a dietary supplement for livestock or humans exposed to DON-contaminated food or feed.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13568-023-01564-5.