Estrogen toxicity has been an area of priority in aquatic toxicology over the last 20 yr. Currently, estrogen toxicity is primarily linked to classical estrogen signaling, the interaction of estrogen receptors alpha and beta (ERα and ERβ). Recent evidence has indicated that a rapid, nongenomic, nonclassical estrogen signaling pathway exists via the G protein-coupled estrogen receptor (GPER), which is expressed in many biological systems, with roles in the cardiovascular system. The objective of the present study was to investigate the effect of 17α-ethinylestradiol (EE2) on the heart rate of embryonic Japanese medaka (Oryzias latipes). A significant decrease (bradycardia) in embryonic heart rate was observed at all treatment concentrations (0.1, 1, 10, 100, and 1000 ng/L EE2) at 144, 168, and 192 h postfertilization (hpf; p ≤ 0.05), whereas 120 and 216 hpf embryos experienced a significant decrease from the control at 10, 100, and 1000 ng/L EE2 and 0.1, 100, and 1000 ng/L EE2, respectively (p ≤ 0.05). In addition, using select estrogen receptor modulators, it was demonstrated that estrogen-induced bradycardia appears to be linked to GPER and not ERα and ERβ. The present study highlights GPER as a novel and alternative mode of action for EE2 toxicity at environmentally relevant concentrations. Environ Toxicol Chem 2020;39:904-912. © 2020 SETAC.