Introduction : Toxoplasma gondii is considered the third most important of 31 foodborne parasites identified in Europe by the FAO/WHO, threatening public health. The development of effective vaccines or drugs against animal and human toxoplasmosis, has been hampered by a lack of data on the sexual reproductive cycle of T. gondii which occurs only in felids. Therefore, we aim to study the interactions of T. gondii with the feline intestinal microenvironment to decipher the local mucosal immune response and understand its impact on parasite life cycle. Methods: Intestinal explants have previously been used to study intestinal pathologies in various species, but until now cat specific models are lacking. Here we report the development of a feline intestinal explant model including both duodenum and ileum tissues. Using this model, we applied RNASCope® technology on the intestinal tissue to study cytokine expression in different culture conditions and in presence or not of T. gondii. Results : Explant enterocytes can be infected by T. gondii tachyzoites within 24h of infection in vitro in hyperoxic conditions and host cytokine responses can be detected. However, incubation in these hyperoxic conditions induced variations in both Th1 and Th2 cytokine expression. Discussion/Conclusion : These preliminary results validate the use of intestinal explants and the RNASCope® technology to study T. gondii interactions with the feline intestinal mucosa and the immune response of the definitive host. A better understanding of these species-specific interactions will help develop vaccine strategies for improved animal and public health.