Gefitinib is a tyrosine kinase inhibitor (TKI) that selectively inhibits the epidermal growth factor receptor (EGFR), hampering cell growth and proliferation. Due to its action, gefitinib has been used in the treatment of cancers that present abnormally increased expression of EGFR. However, side effects from gefitinib therapy may occur, among which diarrhoea is most common, that can lead to interruption of the planned therapy in the more severe cases. The mechanisms underlying intestinal toxicity induced by gefitinib are not well understood. Therefore, this study aims at providing insight into these mechanisms based on transcriptomic responses induced in vitro. A 3D culture of healthy human colon and small intestine (SI) organoids was exposed to 0.1, 1, 10 and 30 µM of gefitinib, for a maximum of three days. These drug concentrations were selected using physiologically-based pharmacokinetic simulation considering patient dosing regimens. Samples were used for the analysis of viability and caspase 3/7 activation, image-based analysis of structural changes, as well as RNA isolation and sequencing via high-throughput techniques. Differential gene expression analysis showed that gefitinib perturbed signal transduction pathways, apoptosis, cell cycle, FOXO-mediated transcription, p53 signalling pathway, and metabolic pathways. Remarkably, opposite expression patterns of genes associated with metabolism of lipids and cholesterol biosynthesis were observed in colon versus SI organoids in response to gefitinib. These differences in the organoids' responses could be linked to increased activated protein kinase (AMPK) activity in colon, which can influence the sensitivity of the colon to the drug. Therefore, this study sheds light on how gefitinib induces toxicity in intestinal organoids and provides an avenue towards the development of a potential tool for drug screening and development.