Neoadjuvant chemotherapy is often used in the treatment of advanced esophageal cancer. In this study, we determined the impact of chemotherapy on microRNA (miRNA) expression in esophageal cancer cells, and whether identified changes might have biological relevance. Two esophageal carcinoma cell lines (one adenocarcinoma and one squamous cell carcinoma) were treated with cisplatin or 5-fluorouracil for 24 or 72 h. RNA was extracted from cells following 24-h treatment, and used for microarray studies. Promising miRNA candidates were selected for RT-PCR validation. Target prediction using TargetScan, combined with bioinformatic analysis (Ingenuity Pathway Analysis, IPA), was performed to evaluate the implications of the altered miRNA expression. Thirteen miRNAs (miR-199a-5p, miR-302f, miR-320a, miR-342-3p, miR-425, miR-455-3p, miR-486-3p, miR-519c-5p, miR-548d-5p, miR-617, miR-758, miR-766, miR-1286) were deregulated after 24- and/or 72-h treatment in both cell lines, and most miRNAs presented similar expression changes after short- or long-term exposure. IPA revealed that the major networks which incorporate the predicted targets, include functions such as 'Cell death', 'Cell cycle', 'Cellular growth and proliferation', 'DNA replication, recombination, and repair' and 'Drug metabolism'. Cisplatin or 5-fluorouracil alter miRNA expression in esophageal cancer cells. IPA suggests that these miRNAs may target molecular pathways involved in cell survival after chemotherapy.