Enzymes used for passaging human pluripotent stem cells (hPSCs) digest cell surface proteins, resulting in cell damage. Moreover, cell dissociation using divalent cation-free solutions causes apoptosis. Here we report that Mg(2+) and Ca(2+) control cell-fibronectin and cell-cell binding of hPSCs, respectively, under feeder- and serum-free culture conditions without enzyme. The hPSCs were detached from fibronectin-, vitronectin- or laminin-coated dishes in low concentrations of Mg(2+) and remained as large colonies in high concentrations of Ca(2+). Using enzyme-free solutions containing Ca(2+) without Mg(2+), we successfully passaged hPSCs as large cell clumps that showed less damage than cells passaged using a divalent cation-free solution or dispase. Under the same conditions, the undifferentiated and early-differentiated cells could also be harvested as a cell sheet without being split off. Our enzyme-free passage of hPSCs under a serum- and feeder-free culture condition reduces cell damage and facilitates easier and safer cultures of hPSCs.