Growing evidence shows that miRNAs play essential roles in cell development by regulating the growth and differentiation of cell lineages. However, the potential roles of miRNA-mediated regulation in the environmental adaptation of organisms are largely unknown. To examine this potential regulatory capability, we systematically identified and compared the miRNAs of zebrafish brains under normal and cold-acclimated conditions using Solexa sequencing. We then predicted the potential target protein genes of the miRNAs and compared their gene expression profile patterns derived from microarray analyses. We defined a set of 25 differentially expressed miRNAs between the cold-acclimatized and normal zebrafish and predicted their functions based on the GO annotation of their targets. We also compared the expression of mRNAs and found that genes related to mRNA processing and response to stress were overrepresented among the up-regulated genes of the cold-stress condition. We analyzed the potential regulatory roles of these miRNAs on gene transcription patterns under low-temperature conditions using several statistical models and a novel, network-based approach. Our results indicate that these miRNAs, either individually or together and either directly or indirectly mediated by transcription factors (TFs), contribute a minor amount to the change in gene expression patterns under low-temperature conditions.