The cold tolerance acquired from acclimation at mild low temperature was characterized in various species, but signal transduction mechanisms underlying the development of cold acclimation remain largely unknown. Here, we deciphered early signaling events associated with immediate temperature drops in zebrafish. Early alternations in the phosphoproteome of larval zebrafish were captured during cold exposure through an optimized sampling protocol and a tandem mass tagging (TMT)-based quantitative phosphoproteomic approach.