MicroRNAs play essential roles in many cellular processes. However, limited information is available regarding the gene structure and transcriptional regulation of miRNAs. We explored the gene cluster encoding miR-433/127 in mammalian species using bioinformatics and in vitro “gene” expression approaches. Multiple sequence alignments (MSA) showed that the precursors of miR-433 and of miR-127 exhibited 95% and 100% similarity, respectively, in human, chimpanzee, horse, dog, monkey, rat, cow, and mouse. MSA of the promoter sequences of miR-433 and of miR-127 revealed lower sequence similarity among these mammalian species. However, the distance between miR-433 and miR-127 was strikingly similar, which was between 986 and 1007 bp and the position of transcription factor (TF) binding motifs, including estrogen related receptor response element (ERRE), was well conserved. Transient transfection assays showed that promoters of miR-433 and of miR-127 from human, rat, and dog were activated by estrogen related receptor gamma (ERRγ) and inhibited by small heterodimer partner (SHP). ChIP assays confirmed the physical association of ERRγ with the endogenous promoters of miR-433 and miR-127. In vitro over-expression of the human, rat, or dog miR-433/127 loci in cells, using an expression vector containing miR-433/127 and their promoter regions, markedly induced a differential expression of both primary and mature miR-433 and miR-127, indicating that miR-433 and miR-127 were possessed from their independent promoters. Our studies for the first time demonstrate a conserved gene structure and transcriptional regulation of miR-433 and miR-127 in mammals. The data suggest that the miR-433/127 loci may have evolved from a common gene of origin.