It has been increasingly clear that changes in gene regulation play important roles in physiological and phenotypic evolution. Rewiring gene-regulatory networks, i.e., alteration of the gene-regulation system for different biological functions, has been demonstrated in various species. Posttranscriptional regulons have prominent roles in coordinating gene expression in a variety of eukaryotes. In this study, using Puf4p in fungi as an example, we demonstrate that posttranscriptional regulatory networks can also be rewired during evolution. Although Puf4p is highly conserved in fungi, targets of the posttranscriptional regulon are functionally diverse among known fungal species. In the Saccharomycotina subdivision, target genes of Puf4p mostly conduct function in the nucleolus; however, in the Pezizomycotina subdivision, they are enriched in the mitochondria. Furthermore, we demonstrate different regulation efficiencies of mitochondrial function by PUF proteins in different fungal clades. Our results indicate that rewiring of posttranscription regulatory networks may be an important way of generating genetic novelties in gene regulation during evolution.