Host defense peptides (HDPs) possess direct antibacterial, antineoplastic, and immunomodulatory abilities, playing a vital role in innate immunity. Dietary-regulated HDP holds immense potential as a novel pathway for preventing infection. In this study, we examined the regulation mechanism of HDPs (pEP2C, pBD-1, and pBD-3) and cytokines (IL-8 and IL-18) expression by sodium phenylbutyrate (PBA). The effects of PBA on HDP induction and the mechanism involved were studied in porcine intestinal epithelial cell lines (IPEC J2). In this study, the results showed that HDPs (pEP2C, pBD-1, and pBD-3) and cytokines (IL-8 and IL-18) expression was increased significantly upon stimulation with PBA in IPEC J2 cells. Furthermore, toll-like receptor 2 (TLR2) and TLR4 were required for the PBA-mediated upregulation of the HDPs. This process occurred and further activated the NF-κB pathway via the phosphorylation of p65 and an IκB α synthesis delay. Meanwhile, histone deacetylase (HDAC) inhibition and an increased phosphorylation of histone H3 on serine S10 also occurred in PBA-induced HDP expression independently with TLR2 and TLR4. Furthermore, p38-MAPK suppressed PBA-induced pEP2C, pBD-1 pBD-3, IL-8, and IL-18 expression, but ERK1/2 failed to abolish the regulation of pBD-3, IL-8, and IL-18. Moreover, epidermal growth factor receptor (EGFR) is involved in PBA-mediated HDP regulation. We concluded that PBA induced HDP and cytokine increases but did not cause an excessive pro-inflammatory response, which proceeded through the TLR2 and TLR4-NF-κB pathway and histone modification in IPEC J2 cells.