Glioblastoma multiforme (GBM) is the most common and aggressive form of brain cancer. Evidences have suggested that CD133 is a marker for a subset of glioblastoma cancer stem cells. However, whether miRNA plays a critical role in CD133 + GBM is poorly understood. Here, we identified that miR‐154 was upregulated in CD133 + GBM cell lines. Knockdown of miR‐154 remarkably suppressed proliferation and migration of CD133 + GBM cells. Further study found that PRPS1 was a direct target of miR‐154 in CD133 + GBM cells. Overexpression of PRPS1 exhibited similar effects as miR‐154 knockdown in CD133 + GBMs. Our study identified miR‐154 as a previously unrecognized positive regulator of proliferation and migration in CD133 + GBM cells and a potentially therapeutic target of GBMs. Copyright © 2016 John Wiley & Sons, Ltd.