BACKGROUND/AIM: Alterations of microRNA expression in three-dimensional spheroids were examined to identify novel microRNAs that might be associated with tumorigenesis. MATERIALS AND METHODS: Using microRNA microarray analysis, we screened for microRNAs that were dramatically up-regulated inside three-dimensional spheroids in genetically-modified HCT116 human colon cancer cells expressing Copepoda Green Fluorescent Protein under hypoxia. RESULTS: miR-3148 was identified as a possible candidate onco-microRNA. A growth advantage of HCT116 cells stably expressing miR-3148 (HCT116-miR3148) was observed compared to parental cells in vivo, but not in vitro. Additionally, no change in growth under hypoxic or starvation conditions was seen in these cells cultured two-dimensionally; however, HCT116-miR3148 cells maintained as three-dimensional spheroids were highly resistant to hypoxic conditions. HCT116-miR3148 cells were more sensitive to mitogen-activated protein kinase (MAPK) kinase inhibitors and extracellular signal-regulated kinase (ERK) inhibitors. CONCLUSION: MiR-3148 may be a novel onco-microRNA that protects cancer cells from serious stress conditions through the MAPK/ERK pathway, especially in vivo.