Deoxynivalenol (DON) is the most common mycotoxin that frequently contaminates human food and animal feed, resulting in intestinal diseases and systemic immunosuppression. Glycyrrhinic acid (GA) exhibits various pharmacological activities. To investigate the protective mechanism of GA for DON-induced inflammation and apoptosis in IPEC-J2 cells, RNA-seq analysis was used in the current study. The IPEC-J2 cells were treated with the control group (CON), 0.5 μg/mL DON, 400 μg/mL GA, and 400 μg/mL GA+0.5 μg/mL DON (GAD) for 6 h. Results showed that 0.5 μg/mL DON exposure for 6 h could induce oxidative stress, inflammation, and apoptosis in IPEC-J2 cells. GA addition could specifically promote the proliferation of DON-induced IPEC-J2 cells in a dose- and time-dependent manner. In addition, GA addition significantly increased Bcl-2 gene expression (
P
<
0.05
) and superoxide dismutase and catalase activities (
P
<
0.01
) and decreased lactate dehydrogenase release, the contents of malonaldehyde, IL-8, and NF-κB (
P
<
0.05
), the relative mRNA abundances of IL-6, IL-8, TNF-α, COX-2, NF-κB, Bax, and caspase 3 (
P
<
0.01
), and the protein expressions of Bax and TNF-α. Moreover, a total of 1576, 289, 1398, and 154 differentially expressed genes were identified in CON vs. DON, CON vs. GA, CON vs. GAD, and DON vs. GAD, respectively. Transcriptome analysis revealed that MAPK, TNF, and NF-κB signaling pathways and some chemokines played significant roles in the regulation of inflammation and apoptosis induced by DON. GA may alleviate DON cytotoxicity via the TNF signaling pathway by downregulating IL-15, CCL5, and other gene expressions. These results indicated that GA could alleviate DON-induced oxidative stress, inflammation, and apoptosis via the TNF signaling pathway in IPEC-J2 cells.