MicroRNAs (miRNAs) are small noncoding RNA and play an essential role in gene regulation. In this study, we investigated regulation of gene expression during neuronal differentiation of mouse P19 embryonic carcinoma cells and described a systematic pathway of gene regulation involving miRNAs. In the pathway, downregulation of Lin28 involved in blocking the let-7 maturation and upregulation of let-7 occur following induction of the differentiation, thereby triggering suppression of the downstream High Mobility Group A2 (Hmga2) gene expression via activation of gene silencing mediated by let-7. Our data further suggest that miR-9, as well as miR-125b, participate in the reduction of the Lin28 expression. The gene regulation involving miRNAs likely contributes to a rapid and programmed change in gene expression in neuronal differentiation of P19 cells.