Mastitis is one of the most common diseases of dairy cattle and can be caused by physical stress, chemicals and microbial infection. Staphylococcus aureus is the most common pathogens that induce mastitis in dairy cattle. In this study, bovine mammary epithelial cells (BMECs) were treated either with lipoteichoic acid (LTA, 30 µg/ml) or 1 × phosphate‐buffer saline (PBS, control) and RNA‐Seq was applied to explore the effect of LTA on the expression microRNAs (miRNAs) in BMECs. Compared to the control group, 43 miRNAs were significantly up‐regulated and eight miRNAs were significantly down‐regulated. Additionally, 724 genes were significantly up‐regulated and 13 genes were significantly down‐regulated in LTA group relative to the control group. Bta‐miR‐196a, bta‐miR‐2285aj‐5p, bta‐miR‐143, bta‐miR‐2433, bta‐miR‐2284f and bta‐miR‐2368‐3p were selected from 51 differentially expressed miRNAs and are discussed in this manuscript. Target gene prediction revealed that the target genes of these six miRNAs were all differentially expressed, including MT1E, SPDYA, FGL1, TLR2, PAPOLG, ZDHHC17 and SMC4. Subsequently, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the target genes with differentially expressed miRNAs were enriched in mitogen‐activated protein kinase (MAPK) signalling pathway, rheumatoid arthritis and cancer. Therefore, the results of this study provided new evidences for the molecular mechanism of LTA‐induced mastitis, which may provide new targets for the diagnosis and treatment of mastitis in dairy cattle.