Estrogens act by binding to estrogen receptors alpha and beta (ERα, ERβ), ligand-dependent transcription factors that play crucial roles in sex differentiation, tumor growth and cardiovascular physiology. Estrogens also activate the G protein-coupled estrogen receptor (GPER), however the function of GPER in vivo is less well understood. Here we find that GPER is required to maintain normal heart rate in zebrafish embryos. Acute exposure to estrogens increased heart rate in wildtype and in ERα and ERβ mutant embryos but not in GPER mutants. Nuclear estrogen receptor signaling remained normal in GPER mutant embryos, however GPER mutant embryos exhibited reduced basal heart rate while heart rate was normal in ERα and ERβ mutants. We detected GPER transcript in discrete regions of the brain but not in the heart. In the brain, we observed gper expression in cells lacking nuclear estrogen receptor activity, suggesting that GPER acts in the brain to regulate heart rate independently of nuclear estrogen receptor signaling. Our results demonstrate that estradiol plays a previously unappreciated role in the acute modulation of heart rate during zebrafish embryonic development and that GPER functions as an autonomous estrogen receptor in vivo to regulate basal heart rate.