Controllable regulation of cell behavior is one of the most important factors conducive to the restoration of tissue functions. Recently, various strategies have been developed using physical or chemical cues. Although these techniques are effective, the high cost and complex fabrication procedures impede their application. In this study, we used a low cost and simple strategy to fabricate PVA/silk fibroin composite hydrogels using a cyclic freeze-thaw method. With the increase of freezethaw cycles, the pore size of hydrogels decreased, the elastic modulus increased, and the swelling rate decreased. Furthermore, we chose two shapes of model cells, a spindle using bone marrow mesenchymal stem cells and smooth muscle cells, and a round shape using BV2 microglial cells. PVA/ silk fibroin composite hydrogels inhibited the adhesion and proliferation of stem cells and muscle cells and changed their cell shape from spindle to round, maintained the initial round shape of BV2 microglial cells, and promoted the proliferation of BV2 microglial cells. These results demonstrate that PVA/silk fibroin composite hydrogels can be used as a novel hydrogel system to regulate cell behavior.