Simple SummaryLow temperature is an often overlooked stress that many fish face due to both natural and anthropogenic causes. Japanese flounder (Paralichthys olivaceus) is an economically important aquaculture fish species broadly cultivated in east Asia, mainly along the coast of the Bohai and Yellow Seas in China, as well as the coast of Korea and east Japan. Natural and cultivatedP. olivaceusmay suffer from cold stress during the winter months. In this study, modulated transcriptomic responses to 10 °C acute cold stress were investigated in the gills, hearts, livers, and spleens ofP. olivaceus. Based on transcriptome and weighted gene coexpression network analysis, tissue-specific cold responsive modules (CRMs) were identified, which revealed a cascade of specific cellular responses to cold stress in different tissues. Our results illustrate the diverse and modulated regulation of the cellular process and stress response to low temperature, which provide essential insights for the conservation and cultivation ofP. olivaceusin cold water.AbstractLow temperature is among the important factors affecting the distribution, survival, growth, and physiology of aquatic animals. In this study, coordinated transcriptomic responses to 10 °C acute cold stress were investigated in the gills, hearts, livers, and spleens of Japanese flounder (Paralichthys olivaceus), an important aquaculture species in east Asia. Histological examination suggested different levels of injury amongP. olivaceustissues after cold shock, mainly in the gills and livers. Based on transcriptome and weighted gene coexpression network analysis, 10 tissue-specific cold responsive modules (CRMs) were identified, revealing a cascade of cellular responses to cold stress. Specifically, five upregulated CRMs were enriched with induced differentially expressed genes (DEGs), mainly corresponding to the functions of “extracellular matrix”, “cytoskeleton”, and “oxidoreductase activity”, indicating the induced cellular response to cold shock. The “cell cycle/division” and “DNA complex” functions were enriched in the downregulated CRMs for all four tissues, which comprised inhibited DEGs, suggesting that even with tissue-specific responses, cold shock may induce severely disrupted cellular functions in all tissues, reducing aquaculture productivity. Therefore, our results revealed the tissue-specific regulation of the cellular response to low-temperature stress, which warrants further investigation and provides more comprehensive insights for the conservation and cultivation ofP. olivaceusin cold water.