Patulin (PAT) is a mycotoxin widely found in fruits and vegetables. Several reviews and studies have hypothesized that in vivo PAT toxicity is related to gut barrier dysfunction, but evidence for this is not substantial. The goal of the present study was to further demonstrate the role of the gut barrier in food-borne PAT toxicity. In vitro assays showed that PAT exposure induced significant cell death, inhibited the mRNA expressions of tight junction proteins and increased gut permeability in Caco-2 cell monolayers. An acute PAT exposure animal trial reported for the first time an association between PAT-induced disruption of the gut barrier and endotoxemia in mice. Sub-chronic PAT exposure also inhibited the expression of ZO-1 in the gut and induced both intestinal and systematic inflammation in mice. Dietary supplements with previously reported protective effects on the gut barrier, such as docosahexaenoic acid and Lactobacillus plantarum CCFM8610, were able to recover the PAT-induced gut barrier dysfunction and significantly alleviate PAT toxicity in vivo. Another L. plantarum strain, CCFM11, with poor gut barrier modulation ability, failed to exhibit identical protective effects against PAT toxicity to L. plantarum CCFM8610. Our results indicated that PAT-induced disruption of the gut barrier and bacterial translocation may be another toxic mechanism of PAT besides its inherent cytotoxicity. Gut barrier protection may be considered an important target for the prevention of PAT toxicity.