Background <p>Acute respiratory distress syndrome (ARDS) is a heterogeneous condition with varying response to prone positioning. We aimed to identify subphenotypes of ARDS patients undergoing prone positioning using machine learning and assess their association with mortality and response to prone positioning.</p> Methods <p>In this retrospective observational study, we enrolled 353 mechanically ventilated ARDS patients who underwent at least one prone positioning cycle. Unsupervised machine learning was used to identify subphenotypes based on respiratory mechanics, oxygenation parameters, and demographic variables collected in supine position. The primary outcome was 28-day mortality. Secondary outcomes included response to prone positioning in terms of respiratory system compliance, driving pressure, PaO₂/FiO₂ ratio, ventilatory ratio, and mechanical power.</p> Results <p>Three distinct subphenotypes were identified. Cluster 1 (22.9% of whole cohort) had a higher PaO₂/FiO₂ ratio and lower Positive End-Expiratory Pressure (PEEP). Cluster 2 (51.3%) had a higher proportion of COVID-19 patients, lower driving pressure, higher PEEP, and higher respiratory system compliance. Cluster 3 (25.8%) had a lower pH, higher PaCO₂, and higher ventilatory ratio. Mortality differed significantly across clusters (p = 0.03), with Cluster 3 having the highest mortality (56%). There were no significant differences in the proportions of responders to prone positioning for any of the studied parameters. Transpulmonary pressure measurements in a subcohort did not improve subphenotype characterization.</p> Conclusions <p>Distinct ARDS subphenotypes with varying mortality were identified in patients undergoing prone positioning; however, predicting which patients benefited from this intervention based on available data was not possible. These findings underscore the need for continued efforts in phenotyping ARDS through multimodal data to better understand the heterogeneity of this population.</p>