MicroRNAs (miRNAs) are small noncoding RNA molecules produced by miR genes which are able to control the expression of a large number of cellular proteins by targeting mRNAs of protein coding genes. It has been suggested that modification of miR gene expression could be an important factor in the development and maintenance of the neoplastic state. It is also reasonable to hypothesize that antineoplastic drugs could be able to alter miR gene expression pattern since most of them are able to interfere with nucleic acid metabolism and gene expression. Here we show that 5-fluorouracil (5-FU), a classical antimetabolite largely used in the clinic, is able to change significantly the expression of several miR genes. In colon cancer cells, at a clinically relevant concentration, the drug up-regulates or down-regulates in vitro the expression of 19 and 3 miR genes, respectively, by a factor of not less than two-fold. In some instances, 5-FU up-regulates miR genes that are already over-expressed in neoplastic tissues, including, for example, miR-21 that is associated with anti-apoptotic functions characterizing malignant cells. In this case, it is possible that drug-induced miR gene dysregulation could be the expression of cellular response to the toxic effects of the agent. On the contrary, in other instances the drug influences the expression of miR genes in a direction that is opposite to that induced by neoplastic transformation. A typical example is provided by miR-200b, that is up-regulated in various tumors and down-regulated by treatment with the antimetabolite. Noteworthy, it is known that miR-200b suppresses a gene that codes for a protein tyrosine phosphatase (PTPN12) that inactivates products of oncogenes, such as c-Abl, Src or Ras. In conclusion, the present results support the hypothesis that 5-FU can alter profoundly miR gene expression pattern. This effect could be responsible, at least in part, of the multi-target pleiotropic influence manifested by the drug on malignant cells.