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Abscisic acid (ABA) is an important plant hormone that mediates abiotic stresses in plant growth and development. A number of E3 ligases have been reported to be involved in ABA signaling pathway. In this study, we identified a C3H2C3 RING-type E3 ligase, Arabidopsis thaliana Tόxicos en Levadura 61 (ATL61), which regulated drought stress in planta. Enzyme assay in vitro demonstrated that ATL61 had E3 ubiquitin ligase activity, while point mutation of ATL61H109A, H122A (mATL61) abolished its E3 ubiquitin ligase activity. ATL61 overexpression plants exhibited ABA hypersensitivity and were more tolerant to drought, while the atl61 mutant plants were insensitive to ABA. Moreover, mATL61 overexpression lines exhibited similar ABA-related phenotypes with wild type (WT) plants. The transcript abundances of ABA-mediated drought stress-related genes RD20 and RD22 were higher in ATL61 overexpression plants than those in WT, atl61, and mATL61 plants. Our results indicated that ATL61 acted as a positive regulator in the ABA-mediated drought stress response. • ATL61 is a C3H2C3 RING type E3 ligase with necessary H109 and H122 residues. • ATL61 is induced by ABA and other abiotic stresses and involved in ABA-mediated plants tolerance to drought. • E3 ligase activity of ATL61 was critical to the function of ATL61 in ABA signaling pathway. [ABSTRACT FROM AUTHOR]