Stem cell therapy represents one of the most promising approaches for tissue repair and regeneration. However, the full potential of stem cell therapy remains to be realized. One major challenge is the insufficient homing and retention of stem cells at the desired sites after delivery. Here, we provide a proof-of-principle demonstration of magnetic targeting and retention of human muscle-derived stem cells (hMDSCs) through magnetic force-mediated internalization of magnetic iron oxide nanoparticles (MIONs) and the use of a micropatterned magnet. We found that the magnetic force-mediated cellular uptake of MIONs occurs through an endocytic pathway, and the MIONs were exclusively localized in the lysosomes. The intracellular MIONs had no detrimental effect on the proliferation of hMDSCs or their multilineage differentiation, and no MIONs were translocated to other cells in a coculture system. Using hMDSCs and three other cell types including human umbilical vein endothelial cells (HUVECs), human dermal fibroblasts (HDFs), and HeLa cells, we further discovered that the magnetic force-mediated MION uptake increased with MION size and decreased with cell membrane tension. We found that the cellular uptake rate was initially increased with MION concentration in solution and approached saturation. These findings provide important insight and guidance for magnetic targeting of stem cells in therapeutic applications.