MicroRNAs (miRNAs) are an extensive class of small non-coding RNAs with important roles in the regulation of gene expression. Little study has been performed on identifying miRNAs in Bombyx mori (silkworm). Here, 41 conserved miRNAs were identified by a computational homology search approach and some of them were selected and confirmed experimentally. Some characteristics such as position-specific nucleotide distribution, evolutionary conservation and clustered organization have been described. Through general method of miRNA target identification, we also find that at least 4 miRNAs (miR-33/-190/-276/-7) target fibroin L chain transcripts, suggesting that miRNAs might play an important role in the regulation of silk protein production. In order to validate this, 3'-UTR of fibroin L chain mRNA was cloned and several expression vectors were constructed. Assays of semi-RT-PCR, histochemical and biochemical assays after agroinfiltration indicated that miRNAs can repress reporter gene (GUS) expression with sequence complement. Clearly, agroinfiltration proved a highly efficient method to identify the function of miRNAs inhibiting their targets mRNA in heterologous system.