Cryogel has macroporous structure and advantages of mechanical stability and injectability for biomedical applications. Three-dimensional (3D) printing is a customized manufacturing technology. However, there is little research on 3D printing of cryogel. In this work, we developed a 3D-printable chitosan cryogel using difunctional polyurethane nanoparticles as the crosslinker that reacted with chitosan at 4 °C for 4 h to form a stable feeding hydrogel (pre-cryogel) for 3D printing. The printed pre-cryogel was frozen at -20 °C to form 3D-printed chitosan cryogel. The 3D-printed cryogel had properties similar to those of bulk cryogel such as high compressibility, elastic recovery, and water absorption (≈3200%). Results from cell experiments indicated that the 3D-printed chitosan cryogel scaffolds provided good mechanical integrity for proliferation and chondrogenic differentiation of human adipose-derived adult stem cells. The 3D-printed chitosan cryogel scaffolds with injectability and shape recovery property are potential biomaterials for customized tissue engineering and minimally invasive surgery.