Estradiol-17beta (E₂) maintains high cAMP levels and meiotic arrest in zebrafish oocytes through activation of G protein-coupled estrogen receptor (GPER). The catecholestrogen 2-hydroxyestradiol-17beta (2-OHE₂) has an opposite effect to that of E₂ on oocyte maturation (OM) and cAMP levels in Indian catfish oocytes. We tested the hypothesis that 2-OHE₂ is produced in zebrafish ovaries and promotes the resumption of oocyte meiosis through its action as a GPER antagonist. Ovarian 2-OHE₂ production by estrogen-2-hydroxylase (EH) was up-regulated by gonadotropin treatment at the onset of OM, consistent with a physiological role for 2-OHE₂ in regulating OM. The increases in EH activity and OM were blocked by treatment with CYP1A1 and CYP1B1 inhibitors. Expression of cyp1a, cyp1b1, and cyp1c mRNAs was increased by gonadotropin treatment, further implicating these Cyp1s in 2-OHE₂ synthesis prior to OM. Conversely, aromatase activity and cyp19a1 mRNA expression declined during gonadotropin induction of OM. 2-OHE₂ treatment significantly increased spontaneous OM in defolliculated zebrafish oocytes and reversed the inhibition of OM by E₂ and the GPER agonist G-1. 2-OHE₂ was an effective competitor of [³H]-E₂ binding to recombinant zebrafish GPER expressed in HEK-293 cells. 2-OHE₂ also antagonized estrogen actions through GPER on cAMP production in zebrafish oocytes, resulting in a reduction in cAMP levels. Stimulation of OM by 2-OHE₂ was blocked by pretreatment of defolliculated oocytes with the GPER antibody. Collectively, the results suggest that 2-OHE₂ functions as a GPER antagonist and promotes OM in zebrafish through blocking GPER-dependent E₂ inhibition of the resumption of OM.