The intestinal epithelium performs vital functions such as nutrient absorption and acting as an intestinal barrier to maintain the host’s homeostasis. Mycotoxin, which affects the processing and storage of animal feedstuff, is a problematic pollutant in farming products. Ochratoxin A generated byAspergillusandPenicilliumfungi causes inflammation, intestinal dysfunction, decline in growth, and reduced intake in porcine and other livestock. Despite these ongoing problems, OTA-related studies in intestinal epithelium are lacking. This study aimed to demonstrate that OTA regulates TLR/MyD88 signaling in IPEC-J2 cells and induces barrier function impairment through tight junction reduction. We measured expression of TLR/MyD88 signaling-related mRNAs and proteins. The indicator of intestinal barrier integrity was confirmed through immunofluorescence and transepithelial electrical resistance. Additionally, we confirmed whether inflammatory cytokines and barrier function were affected by MyD88 inhibition. MyD88 inhibition alleviated inflammatory cytokine levels, tight junction reduction, and damage to barrier function due to OTA. These results indicate that OTA induces TLR/MyD88 signaling-related genes and impairs tight junctions and intestinal barrier function in IPEC-J2 cells. MyD88 regulation in OTA-treated IPEC-J2 cells mitigates the tight junction and intestinal barrier function impairments. Our findings provide a molecular understanding of OTA toxicity in porcine intestinal epithelial cells.