5-Fluorouracil (5-FU) is a classic chemotherapeutic drug that has been widely used for breast cancer treatment. Although aberrant expression of protein-coding genes was observed after 5-FU treatment, the regulatory mechanism remains poorly understood. MicroRNAs (miRNAs) are a newly identified class of small regulatory RNAs which play an important role in gene regulation at the post-transcriptional levels. Recent evidence suggests an important role of miRNAs in initiation, progression, and metastasis of human cancers. In this study, using a combined advanced microarray and quantitative real-time PCR (qRT-PCR) technology, we show that 5-FU significantly alters the global expression profile of miRNAs in vitro. After 48 h of treatment with a low dose (0.01 µM), 42 miRNAs were differentially expressed in MCF-7 breast cancer cell line. Of these, 23 miRNAs were up-regulated with up to 4.59-fold changes, while 19 were down-regulated with up to 1.89-fold changes. A majority of these miRNAs are associated with cancer development, progression, and metastasis. Target prediction and GO analysis suggest that these differentially expressed miRNAs potentially target many oncogenes, tumor suppressor genes and genes related to programmed cell death, activation of immune response, and cellular catabolic processes.