A group of the concentrations of inactivated Staphylococcus aureus(0, 104, 105, 106, 107, 108, 109 cfu/mL) and of peptidoglycan(PG)(0.0, 0.1, 1.0, 10.0 μg/mL) were selected to induce the expression of regenerating protein Ⅲ γ(RegIIIγ) in porcine intestinal epithelial cells(IPEC-J2). The levels of RegIIIγ mRNA and protein were detected by qPCR and western blot assay, and the expression variations of RegⅢγ, P65, P38, C-Jun amino terminal kinase(JNK) and extracellular regulatory protein kinase(ERK) were analyzed to further explore the molecular mechanism of RegⅢγ expression regulation. The results showed that both inactivated S. aureus and PG had inhibitory effects on cellular viability of IPEC-J2. In comparison to the treatment without inactivated S. aureus, the mRNA and protein expression levels of RegⅢγ was higher in the groups of inactivated S. aureus treatment and showed a does-dependent trend, and the mRNA and protein expression levels were significantly increased when the concentration of inactivated S. aureus was 109 cfu/mL. Similarly, PG also induced the expression of RegⅢγ. Compared with the treatment without PG, the mRNA and protein expression levels of RegⅢγ were significantly higher when PG was 1.0 μg/mL. The expression levels of P65, P38, JNK, and ERK in IPEC-J2 were significantly increased by adding 109 cfu/mL inactivated S. aureus or 1.0 μg/mL PG, compared with that without adding inactivated S. aureus or PG. These results suggested that the expression of RegⅢγ in IPEC-J2 might be regulated by two signaling pathways: nuclear transcription factor κB(NF-κB) and mitogen-activated protein kinase(MAPK) downstream of the primary myeloid differentiation response protein MyD88.