Abstract: The purpose of this article was to investigate the bioaccessibility, absorption-transport rate, and cytotoxicity of patulin in apple, pear, orange and tomato using in vitro simulated gastrointestinal digestion and cell models. Firstly, Penicillium expansum was inoculated into fruits and cultured for 7 days. Rotten parts were collected for in vitro dynamic digestion and Caco-2 cell model research. Patulin was analyzed by high performance liquid chromatography, and the bioaccessibility, cell absorption-transport rate were calculated. Finally, the effects of patulin from intestinal digestive juices on cell viability and cytokine expression were investigated. The results revealed that the bioaccessibility of patulin from the four fruit substrates in the gastric and intestinal digestive juices was 11.30%~66.04% and 1.00%~14.52%, respectively. The absorption-transport rates of patulin in Caco-2 cell were all greater than 98%. Cytotoxicity experiments showed that patulin from the intestinal digestive juices of four fruit substrates reduced cell viability to approximately 80%. Additionally, gene expression levels of four proinflammatory factors, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8) were all up-regulated. These findings laid a foundation for the future study of in vivo digestion, absorption and cytotoxicity of patulin in food.