Introduction: Methotrexate (MTX) is a frequently used cytostatic drug in childhood cancer treatment. One of the most severe side effects of this treatment is damage to the highly proliferating intestinal epithelium. This damage can cause therapy adjustment. Aim: To analyze the regulation of intestinal epithelial gene expression during MTX induced intestinal damage. Methods: Balb/C mice were treated with two doses of MTX (120 and 60 mg/kg) on two consecutive days. Six hours, 1, 2, 3 and 9 days after the second injection the mice were sacrificed and the small intestine was collected for histology and quantification assays. The enterocyte specific markers, sucrase isomaltase (SI) and liver fatty acid binding protein (L-FABP), were studied by immunohistochemisty (IHC) and quantified by dot blot analysis. Transcription factors, known to regulate the expression of SI and/or L-FABP; GATA4, HNF1alpha, HNF4gamma, HNF4alpha and CDX2 were studied by IHC. Results: MTX treatment resulted in intestinal epithelial damage that was most severe at day 3. The intestine was virtually completely regenerated at day 9. Epithelial flattening, villus atrophy, loss of crypts and deterioration of epithelial structure characterize the damage. At day 3 after MTX injection we saw a loss of more than 85% of the enterocyte specific SI expression while L-FABP showed no significant change. GATA4, HNF1alpha, HNF4gamma and HNF4alpha showed complete loss of expression at day 3. CDX2 remained visible in the epithelium throughout all stages of damage. At day 1 the staining shifted from the nucleus to the cytoplasm and returned to the nucleus at day 3. Conclusion: Decreased SI expression after MTX-treatment indicates a decreased sucrose metabolism of the enterocyte, whereas the constant L-FABP expression suggests an unaffected fatty acid transport. The effects of MTX on SI can probably be explained by the decreased expression and absence of nuclear localization of the studied transcription factors. Maintenance of the L-FABP protein, even in the absence of the studied transcription factors, suggests that this protein is most likely post-transcriptionally regulated.