Abstract Background Prone position is frequently used in patients with acute respiratory distress syndrome (ARDS), especially during the Coronavirus disease 2019 pandemic. Our study investigated the ability of pulse pressure variation (PPV) and its changes during a tidal volume challenge (TVC) to assess preload responsiveness in ARDS patients under prone position. Methods This was a prospective study conducted in a 25-bed intensive care unit at a university hospital. We included patients with ARDS under prone position, ventilated with 6 mL/kg tidal volume and monitored by a transpulmonary thermodilution device. We measured PPV and its changes during a TVC (ΔPPV TVC 6–8 ) after increasing the tidal volume from 6 to 8 mL/kg for one minute. Changes in cardiac index (CI) during a Trendelenburg maneuver (ΔCI TREND ) and during end-expiratory occlusion (EEO) at 8 mL/kg tidal volume (ΔCI EEO 8 ) were recorded. Preload responsiveness was defined by both ΔCI TREND ≥ 8% and ΔCI EEO 8 ≥ 5%. Preload unresponsiveness was defined by both ΔCI TREND < 8% and ΔCI EEO 8 < 5%. Results Eighty-four sets of measurements were analyzed in 58 patients. Before prone positioning, the ratio of partial pressure of arterial oxygen to fraction of inspired oxygen was 104 ± 27 mmHg. At the inclusion time, patients were under prone position for 11 (2–14) hours. Norepinephrine was administered in 83% of cases with a dose of 0.25 (0.15–0.42) µg/kg/min. The positive end-expiratory pressure was 14 (11–16) cmH 2 O. The driving pressure was 12 (10–17) cmH 2 O, and the respiratory system compliance was 32 (22–40) mL/cmH 2 O. Preload responsiveness was detected in 42 cases. An absolute change in PPV ≥ 3.5% during a TVC assessed preload responsiveness with an area under the receiver operating characteristics (AUROC) curve of 0.94 ± 0.03 (sensitivity: 98%, specificity: 86%) better than that of baseline PPV (0.85 ± 0.05; p = 0.047). In the 56 cases where baseline PPV was inconclusive (≥ 4% and < 11%), ΔPPV TVC 6–8 ≥ 3.5% still enabled to reliably assess preload responsiveness (AUROC: 0.91 ± 0.05, sensitivity: 97%, specificity: 81%; p < 0.01 vs. baseline PPV). Conclusion In patients with ARDS under low tidal volume ventilation during prone position, the changes in PPV during a TVC can reliably assess preload responsiveness without the need for cardiac output measurements. Trial registration: ClinicalTrials.gov (NCT04457739). Registered 30 June 2020 —Retrospectively registered, https://clinicaltrials.gov/ct2/show/record/NCT04457739