•Esketamine 0.25 mg/kg was noninferior to ketamine 0.5 mg/kg in promoting remission of major depression symptoms 24 h after a single intravenous administration in subjects with treatment-resistant depression.•Esketamine and ketamine demonstrated a similar safety pattern and were well tolerated by most participants.•This is the first head-to-head randomized clinical trial comparing racemic ketamine and esketamine in patients with treatment-resistant depression. Ketamine and its enantiomers have recently been highlighted as one of the most effective therapeutic options in refractory depression. However, racemic ketamine and esketamine have not been directly compared. The aim of this study is to assess the efficacy and safety of esketamine compared to ketamine in patients with treatment-resistant depression (TRD). This is a randomized, double-blind, active-controlled, bicentre, non-inferiority clinical trial, with two parallel groups. Participants were randomly assigned to a 40-min single intravenous infusion of ketamine 0.5 mg/kg or esketamine 0.25 mg/kg. The primary outcome was the difference in remission rates for depression 24 h following intervention using the Montgomery-Åsberg Depression Rating Scale (MADRS), with a non-inferiority margin of 20%. 63 subjects were included and randomly assigned (29 to receive ketamine and 34 to receive esketamine). At 24 h, 24.1% of participants in the ketamine group and 29.4% of participants in the esketamine group showed remission, with a difference of 5.3% (95% CILB -13.6%), confirming non-inferiority. MADRS scores improved from 33 (SD 9.3) to 16.2 (SD 10.7) in the ketamine group and from 33 (SD 5.3) to 17.5 (SD 12.2) in the esketamine one, with a difference of -5.27% (95% CILB, -13.6). Both groups presented similar mild side effects. Esketamine was non-inferior to ketamine for TRD 24 h following infusion. Both treatments were effective, safe, and well tolerated. Registered in Japan Primary Registries Network: UMIN000032355.