Highly porous chitosan microspheres (CSM) were prepared through emulsion-based thermally induced phase separation (TIPS) without using toxic crosslinkers and chemical porogenic agents other than ice. The CSM had an average diameter of ∼150 μm with interconnected pores varying from 20∼50 μm in size. Due to their excellent biocompatibility and unique porous structure, high-performance hepatocyte culture in three-dimensional (3D) space was achieved using the CSM as microcarriers, as cell growth also took place within the internal pores of the CSM, besides their external surface, and multidirectional cell-cell interactions were observed. Enhanced cellular activity and functions were obtained with the CSM microcarriers as compared with 2D cell culture. It is believed that these CSM microcarriers provide a promising platform for 3D cell culture in vitro.