Background: Side population (SP) cells and their relationship to stem cell-like properties have been insufficiently studied in colorectal cancer (CRC). MicroRNAs (miRNAs) have attracted much attention but their roles in the maintenance of SP phenotype remain unclear. Methods: The SPs from CRC cell lines and primary cell cultures were analysed for stem cell-like properties. MiRNA microarray analysis identified miR-328 as a potential stemness miRNA of SP phenotype. The level of miR-328 expression in clinical samples and its correlation with SP fraction were determined. Gain-of-function and loss-of-function studies were performed to examine its roles in cancer stem-like SP cells. Furthermore, bioinformatics prediction and experimental validation were used to identify miR-328 target genes. Results: The SP cells sorted from CRC possess cancer stem cell (CSC)-like properties, including self-renewal, differentiation, resistance to chemotherapy, invasive and strong tumour formation ability. MiR-328 expression was significantly reduced in SP cells compared with Non-SP cells (P<0.05). Moreover, miR-328 expression was downregulated in CRC (n=33, P<0.05) and low miR-328 expression tend to correlate with high SP fraction (n=15, r=0.6559, P<0.05, Pearson's correlation). Functional studies indicated that miR-328 expression affects the number of SP cells. In addition, miR-328 overexpression reversed drug resistance and inhibited cell invasion of SP cells. Furthermore, luciferase reporter assay demonstrated that miR-328 directly targets ABCG2 and MMP16 and affects the levels of mRNA and protein expression in SP cells. Conclusion: These findings indicate that CRC contain cancer stem-like SP cells. MiR-328 has an important role in maintaining cancer stem-like SP phenotype that may be a potential target for effective CRC therapy.