Background: MicroRNAs (miRNAs) have been implicated in the regulation of milk protein synthesis and development of mammary gland (MG).However, the function of miRNAs in regulating lactation is unclear.Therefore, the elucidation of miRNA expression profiles in MG provides a crucial entry into the understanding of the mechanisms of lactation.Methods: In this study, parenchymal tissues were sampled fron the MG of non-and lactating Holstein dairy cows.Purified small RNA was sequenced by Solexa sequencing and processed using ACGT101-miR program and UNAFold software.The IDEG 6 was used to identify differentially expressed miRNAs.A custom-designed nicroarray assay including 523 miRNA probes was performed to analyze miRNA expression patterns.The EGAN software was used to construct network between miRNAs, target genes and lactation.Results: Two miRNA libraries were constructed and sequenced.There were 885 pre-miRNAs encoding for 921 miRNAs, of which 884 were unique and 544 (61.5%) were expressed in both periods.Among them, 284 were known as bovine miRNAs registered in the miRbase database ; 96 miRNAs were conserved among other mammalian ; and 566 miRNAs were bovine-novel candidates.A custom-designed microarray assay was performed to analyze miRNA expression patterns in the MG of lactating and non-lactating dairy cows.Compared against non-lactating MG, a total of 226 miRNAs in the lactating MG showed significant differences in expression (P<0.01).Real-time quantitative PCR was used for validation of the sequencing results of selected 32 miRNAs.Over 50% ofmiRNAs were consistent with Solexa sequencing data.The chromosome positions for all 885 pre-miRNAs were found that 800 premiRNAs matched with bta genome, and only 230 pre-miRNAs were grouped into 55 clusters.Using integrative miRNA target-prediction and network-analysis approaches an interaction network of lactation-related miRNAs and their putative targets was constructed.The networks involved 37 miRNAs detected by this study and 15 target genes around the lactation.The possible biological significance attributed to these differences in expression was investigated using a cell-based model.Conclusion: Our study provides a broad view of the bovine MG miRNAs expression profiles characteristics.117 new miRNAs were identified in MG.There were differences in types and expression levels of miRNAs between lactating and non-lactating bovine MG.(rumeemore), systemic predictions atded the identification of lactation-related miRNAs giving insights into the types ofmiRNAs involved in lactation and how they regulate the process .