Developmental toxicity is a critical issue in nanotoxicity. However, very little is known about the effects of graphene oxide (GO, a widely used carbon material) at predicted environmental concentrations on biological development or the specific molecular mechanisms. The present study established that the development of zebrafish embryos exposed to trace concentrations (1-100 μg/L) of GO was impaired because of DNA modification, protein carbonylation and excessive generation of reactive oxygen species (ROS), especially the superoxide radical. Noticeably, there was a nonmonotonic response of zebrafish developmental toxicity to GO at μg/L to mg/L levels. Transcriptomics analysis revealed that disturbing collagen- and matrix metalloproteinase (MMP)-related genes affected the skeletal and cardiac development of zebrafish. Moreover, metabolomics analysis showed that the inhibition of amino acid metabolism and the ratios of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs) contributed to the above developmental toxicity. The present work verifies the developmental toxicity of GO at trace concentrations and illustrates for the first time the specific molecular mechanisms thereof. Because of the potential developmental toxicity of GO at trace concentrations, government administrators and nanomaterial producers should consider its potential risks prior to the widespread environmental exposure to GO.