Ketamine is a non-competitive channel blocker of N-Methyl-D-Aspartate (NMDA) receptor. A single sub-anesthetic dose of ketamine produces rapid (within hours) and long-lasting antidepressant effects in patients who are resistant to other antidepressants. Ketamine is a racemic mixture of S- and R-ketamine enantiomers, with S-ketamine isomer as the more active antidepressant. Here we determined the cryo-EM structures of human GluN1-GluN2A and GluN1-GluN2B NMDA receptors in complex with S-ketamine, glycine and glutamate. Both electron density maps uncovered the binding pocket for S-ketamine in the central vestibule between the channel gate and selectivity filter. Molecular dynamics simulation revealed that S-ketamine displays motions between two distinct locations within the binding pocket. Two amino acids, L642 on GluN2A (homologous L643 on GluN2B) and N616 on GluN1, were identified as key residues forming hydrophobic and hydrogen-bond interactions with ketamine, and mutations at these identified residues led to the reduced potency of ketamine in blocking the NMDA receptor channel activity. These findings provide the structural basis of ketamine binding and action on human NMDA receptors, and pave the way for future development of ketamine-based antidepressants.