Proline is an amino acid acting as an osmoprotector in plants, as it reduces osmotic potential and enhances plant water uptake, especially under drought. Aluminum (Al) toxicity causes inhibition of root growth and low leaf hydration with negative consequences for photosynthetic performance. Thus, plants under Al toxicity could benefit from proline accumulation. Here we investigated whether proline is induced by Al in Pinus sylvestris, an Al-tolerant woody species. Proline and abscisic acid (ABA) concentration, gas exchange rates, stomatal conductance (gs), relative needle water content (RWC), stem water potential (Ψw), biometric data and plant biomass were evaluated in plants grown in nutrient solution with 0, 250, 500, and 1000 μM Al for 42 days. Plant growth, gas exchange rates, RWC and ABA were the same between the four treatments, as expected for an Al-tolerant species. The maintenance of needle hydration throughout the study provides a plausible explanation for the similar gas exchange values observed between Al-treated and untreated plants. Over the course of 7 to 14 days, plants exposed to Al showed a decrease in midday stem water potential (Ψmd), and an increase in proline concentration. Subsequently, both parameters exhibited similar values between treatments until the end of the study. These findings suggest proline may play a role in osmotic adjustment in P. sylvestris exposed to Al.