Prenatal exposure to anesthetics has raised increasing attention about the neuronal development in offspring. Animal models are usually used for investigation. As a new drug, esketamine is the s-isoform of ketamine and is twice as potent as the racemic ketamine with less reported adverse effects. Esketamine is currently being used and become more favorable in clinical anesthesia work, including surgeries during pregnancy, yet the effect on the offspring is unknown. The present study aimed to elucidate the effects of gestational administration of esketamine on neuronal development in offspring, using a rat model. Gestational day 14.5 pregnant rats received intravenous injections of esketamine. The postnatal day 0 (P0) hippocampus was digested and cultured in vitro to display the neuronal growth morphology. On Day 4 the in vitro experiments revealed a shorter axon length and fewer dendrite branches in the esketamine group. The results from the EdU- imaging kit showed decreased proliferative capacity in the subventricular zone (SVZ) and dentate gyrus (DG) in both P0 and P30 offspring brains in the esketamine group. Moreover, neurogenesis, neuron maturity and spine density were impaired, resulting in attenuated long-term potentiation (LTP). Compromised hippocampal function accounted for the deficits in neuronal cognition, memory and emotion. The evidence obtained suggests that the neurobehavioral deficit due to prenatal exposure to esketamine may be related to the decrease phosphorylation of CREB and abnormalities in N-methyl-d-aspartic acid receptor subunits. Taken together, these results demonstrate the negative effect of prenatal esketamine exposure on neuronal development in offspring rats.Graphical AbstractG14.5 esketamine administration influenced the neurobehavior of the offspring in adolescence. Poorer neuronal growth and reduced brain proliferative capacity in late gestation and juvenile pups resulted in impaired P30 neuronal plasticity and synaptic spines as well as abnormalities in NMDAR subunits. Attenuated LTP reflected compromised hippocampal function, as confirmed by behavioral tests of cognition, memory and emotions. This figure was completed on the website of Figdraw.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10571-023-01354-4.