Recent studies have shown that Non-Alcoholic Fatty Liver Disease (NAFLD) pathogenesis could be related to dysfunction of the intestine; however, the molecular mechanisms underlying these dysfunctions have been poorly investigated, because of the lack of a good model for in vitro studying gut physiology and functions in patients with NAFLD. In our previous studies, we developed an intestinal organoid platform to study diabetes and obesity pathophysiology and we showed that intestinal cell dysfunctions were correlated with impaired cell development in intestinal organoids. The present study aimed to develop a human intestinal organoid model in order to analyse gut function in NAFLD patients. Intestinal organoids were isolated from duodenum biopsy of patients with NAFLD and controls. After 7-10 days of culture time-lapse microscopy, RNA extraction, microarray analysis and Real-time PCR were performed. Time-lapse analysis showed differences in growth of NAFLD organoids compared to control. Through microarray method, we analyzed the entire transcriptome expressed in organoids isolated from patients with NAFLD and controls, we found 600 dysregulated transcripts. Bioinformatic analysis showed that dysregulated transcripts were statistically significantly associated with molecular pathways consistent with NAFLD and gut dysfunction such as metabolic pathways (fatty acids and proteins metabolism, ATP production), inflammatory pathways (TGF-beta signaling pathway, IL-17 signaling pathway), cell proliferation and apoptosis pathways (Hippo signaling pathway, MAPK signaling pathway, Ras signaling pathway), pathways regulating pluripotency of stem cells (Wnt signaling pathway, MAPK signaling pathway, TGF-beta signaling pathway). Finally, microarray data were confirmed through Real-Time PCR of selected transcripts. This finding provides a new molecular in vitro model to study the gut-liver axis in patients affected by NAFLD using a small intestinal organoid platform. This study led to the identification of new molecular signaling pathways that could be useful for better treatment of gut dysfunction in patients with NAFLD.