Abstract. Salsinha YCF, Nurbaiti S, Sebastian A, Indradewa D, Purwestri YA, Rachmawari D. 2022. Proline-related gene expressions contribute to physiological changes of East Nusa Tenggara (Indonesia) local rice cultivars during drought stress. Biodiversitas 23: 3573-3583. An osmoregulation response is one of the mechanisms of adaptation to drought stress. Concerning the synthesis and catabolism of proline as an osmoprotectant, this research aims to study the involvement of proline in regulating physiological change in local rice cultivars from Nusa Tenggara Timur (NTT), Indonesia. Five rice cultivars consisting of Ciherang (drought susceptible) and Situ Bagendit (drought tolerant) and three local rice cultivars, namely Gogo Jak (GJ), Kisol Manggarai (KM), and Boawae Seratus Malam (BSM) were used. Drought treatments were carried out using the fraction of transpirable soil water (FTSW) with control (FTSW 1) and severe drought (FTSW 0.2). The upregulation of OsP5CS and OsP5CR as proline synthesis genes in GJ leaves led to higher proline levels. At the KM cultivar, there was an upregulation of OsPRODH related to proline catabolism. Furthermore, OsNHX1, responsible for proline transport, was higher in the GJ cultivar. The presence of proline also correlated with physiological adaptations. Proline accumulation due to the activation of proline-related gene expressions caused less physiological changes and antioxidant activity reduction and has led to maximum water absorption during a severe drought in the BSM cultivar. The analysis showed non-significant decreased levels of chlorophyll-a and increased levels of chlorophyll-b, carotenoids, and anthocyanins of drought-tolerant cultivars lead to higher leaf number, plant height, and tiller number under drought. Meanwhile, varied activity levels of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) were observed in line with the slight increase in H2O2 and malonaldehyde (MDA) levels during severe drought.