Colon cancer is one of the most common cancers in the world. Multidrug resistance is related to poor prognosis of advanced colon cancer. The side population plays an important role in multiple drug resistance (MDR) of colon cancer. MicroRNA biomarkers of the side population of colon cancer is still unknown. In the present study, we aimed to explore miRNA markers of side population (SP) cells of colon cancer. The side population was sorted by flow cytometry. Cell viability was measured using an MTT assay. MicroRNA profiling analysis was performed to compare microRNA expression levels in the SP cells of colon cancer with levels in the non-SP cells of colon cancer. RT-PCR was applied to verify the result obtained from the microRNA profiling analysis. miR-5000-3p, miR-5009-3P and miR-552 were all found to be upregulated in SP cells of the colon cancer cell lines HCT-15, HT-29 and LoVo. RT-PCR confirmed the result from the microRNA profiling analysis. This implied that miR-5000-3p, miR-5009-3P and miR-552 may be potential microRNA biomarkers of the side population in colon cancer, which may provide new specific targets of the side population for the reversal of MDR of colon cancer.