Effects of temperature on biological processes are complex. Diffusion is less affected than the diverse enzymatic reactions that have distinct individual temperature profiles. Hence thermal fluctuations pose a formidable challenge to ectothermic organisms in which body temperature is largely dictated by the ambient temperature. How cells in ectotherms cope with the myriad disruptive effects of temperature variation is poorly understood at the molecular level. Here we show that nucleocytoplasmic posttranslational modification of proteins with O-linked GlcNAc (O-GlcNAc) is closely correlated with ambient temperature during development of distantly related ectotherms ranging from the insect Drosophila melanogaster to the nematode Caenorhabditis elegans to the fish Danio rerio. Regulation seems to occur at the level of activity of the only two enzymes, O-GlcNAc transferase and O-GlcNAcase, that add and remove, respectively, this posttranslational modification in nucleus and cytoplasm. With genetic approaches in D. melanogaster and C. elegans, we demonstrate the importance of high levels of this posttranslational modification for successful development at elevated temperatures. Because many cytoplasmic and nuclear proteins in diverse pathways are O-GlcNAc targets, temperature-dependent regulation of this modification might contribute to an efficient coordinate adjustment of cellular processes in response to thermal change.