The intestine and liver represent a highly interconnected system, this close functional and vascular association has been called the “gut-liver axis”; some studies have highlighted the presence of intestinal alterations in the context of NAFLD, characterized by a severity proportional to the stage of liver disease. Despite these studies, the knowledge of intestinal alterations in NAFLD patients is limited. To date, no study has applied a high-throughput approach to identify perturbated pathways in NASH-associated intestinal dysfunction. There is no literature data concerning the expression profile dysregulation of coding RNAs or noncoding RNAs in intestinal biopsies of NAFLD patients. Furthermore, to date, there is a lack of noninvasive serum biomarkers able to reflect the compromised intestinal barrier structure and function. To identify molecular pathways involved in NASH-related intestinal dysfunction, we analysed the whole transcriptome expressed in biopsies from seven NASH patients versus five control subjects. We identified 880 dysregulated transcripts, among them 103 had NM and NR annotations in the NCBI database. Through bioinformatics analysis, we observed that dysregulated transcripts were statistically associated with various signalling pathways potentially involved in intestinal dysfunction such as inflammatory, metabolic, fibrosis, apoptotic, cellular stress and cell proliferation pathways. To confirm microarray data, through real time PCR assays, we confirmed the dysregulation of DDX56, SFRP5, C1ORF21, BMP8A, BHLEH40, MDN1, NAP1L2 coding RNAs and FLVCR2AS, LOC101927630, LINC00955 noncoding RNAs in a validation cohort of 24 NASH patients versus 20 controls. Validated transcripts at tissue level were analysed also in serum samples to identify non-invasive biomarkers of NASH-related intestinal dysfunction. Among these, only BHLEH40, NAP1L2, SFRP5, C1ORF21, and LOC101927630 were expressed in serum samples. NAP1L2 and LOC101927630 were downregulated in serum samples of NASH patients versus controls.