Crystallization behavior of polylactic acid (PLA) under CO2 is meaningful due to its effect on foaming behavior during CO2 foaming, while present studies mainly concentrated on overall crystallization behavior. In this study, the effect of CO2 on nucleation and spherulite growth of PLA crystals was in situ investigated separately. During saturation, sorption of CO2 in PLA diluted the molecular chain density, thus increasing molecular chain mobility and weakening the interchain interaction, which reduced the amount of crystal nuclei in PLA. On the other hand, the spherulite growth rate reduced at high temperature and increased at low temperature attributed to the enhanced chain mobility under CO2. Thus, overall crystallization was controlled by the spherulite growth rate at low temperature and crystalline nucleation at high temperature under CO2. Besides, CO2 restricted lamella branching, resulting in an increased lamella thickness and z axis size. Current results revealed the effect of tunable molecular chain mobility and the interchain interaction adjusted by CO2 on nucleation and spherulite growth of PLA.