The long non-coding RNAs (lncRNAs) are known to transcriptionally regulate a wide spectrum of diseases. Here, we screened for potentially functional lncRNAs in a mammary epithelial cell model of bovine mastitis by RNA-Seq technology and identified a class of previously undetected mastitis-related lncRNAs. A novel lncRNA was widely expressed in a variety of bovine tissues with diverse relative abundance and had a relatively low expression in mammary tissue. Given its predicted target gene is TUBA1C, we name it lncRNA-TUB. We found a higher expression of lncRNA-TUB in mammary epithelial cells that received a proinflammatory stimulus compared to normal cells. Knockout of lncRNA-TUB by the CRISPR/Cas9 system revealed that it plays crucial roles in the morphological shape, proliferation, migration and β-casein secretion of mammary epithelial cells. In addition, lncRNA-TUB mediates Escherichia coli-induced inflammatory factor secretion and Staphylococcus aureus adhesion to epithelial cells. Our results suggest that the lncRNAs identified here function in bovine mastitis, and that lncRNA-TUB affects the basic biological characteristics and functions of bovine mammary epithelial cells in inflammatory conditions, providing valuable insights into the mechanisms of bovine mastitis.