MicroRNA (miRNA) are highly conserved, noncoding small RNA involved in post-transcriptional gene regulation in a variety of biological processes. To elucidate roles of miRNA in bovine muscle type specification and maintenance, we sought to determine differentially expressed miRNA between semitendinosus (STD) and masseter (MS) muscles from 3 Japanese black cattle by massively parallel sequencing. Differential gene expression of myosin heavy chain (MyHC) isoforms confirmed that STD and MS were MyHC-2x- and MyHC-1-abundant muscles, respectively. In total, 192 known miRNA and 20 potential new bovine miRNA were obtained from the sequencing. The differentially expressed miRNA with more than 2-fold difference in each muscle were identified. In particular, miR-196a and miR-885 were exclusively expressed in STD muscle, which was validated by quantitative reverse transcription-PCR (P=0.045 and P<0.001, respectively), whereas a slow type-directing miR-208b was highly expressed in MS compared with STD (false discovery rate<0.05). In addition, 16 potential novel miRNA were mapped and confirmed for their precursor structures by computational analyses. The results of functional annotation combined with in silico target analysis showed that the predicted target genes of miR-196a/b and miR-885 enriched gene ontology (GO) terms related to skeletal system development and regulation of transcription, respectively. Moreover, GO terms enriched from predicted targets miRNA suggested that STD-abundant- and MS-abundant-miRNA were associated with embryonic body planning and organ/tissue pattern formation, respectively. The present results revealed that the differentially expressed miRNA between the STD and MS muscles may play key roles to determine muscle type-specific tissue formation and maintenance in cattle thorough attenuating putative target genes involved in different developmental events.