Ketamine is widely used in infants and young children for anesthesia, and subanesthetic doses of ketamine make neurons form new protrusions and promote synapse formation. However, the precise pathological mechanisms remain to be elucidated. In this study, we demonstrated that ketamine administration significantly increased dendritic spine density and maturity in rat cortical neurons in vivo and in vitro. Western blot analysis showed that CRMP2 protein expression was significantly increased in cerebral cortex of ketamine group, and phosphorylation levels of CRMP at Thr514 and Ser522 were significantly reduced. Furthermore, overexpression of CRMP2 promoted the growth of cortical neuron processes and dendritic spines. Although the dendritic field was more complex after adding ketamine and the density of dendritic spines increased, there was no statistical difference and no obvious superposition effect was observed. Moreover, both Ser522 mutant construction of CRMP2, GFP-CRMP2-522D, and mcherry-CDK5 showed similar inhibitory effects on neurite outgrowth, which could be rescued by ketamine. The frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) were significantly inhibited when GFP-CRMP2-522D and mCherry-CDK5 were transfected into cortical neurons and this trend could also be rescued by ketamine. In general, this study reveals a new mechanism by which ketamine promotes the growth and development of dendritic spines in developing cortical neurons.