Mastitis is an endemic disease in the dairy industry and causes large economic losses to dairy farmers due to reduced milk yield and quality. Mastitis is inflammation of one or more mammary glands caused by bacterial infection. Lipopolysaccharide (LPS) is a major outer membrane component of gram-negative bacteria and a major endotoxin that elicits strong mammary inflammation. The major objective of this study was to investigate the effects of LPS on milk protein gene expression in mammary epithelial cells (MEC), using HC11, a mouse MEC line, as a model. HC11 cells were cultured with 0–500 µg of LPS for 3–24 h to assess cell viability through MTT assay and relative gene expression via real-time reverse transcription PCR. LPS reduced HC11 cell viability in a dose- and time- dependent manner as doses of ≥250 ug/mL reduced cell viability significantly at 4 h, but the threshold decreased to ≥100 ug/mL at 24 h (P < 0.05). However, as little as 0.1 µg/mL of LPS dramatically induced mRNA expression of inflammation markers IL-6, IL-1β and TNFα at 3 h and 24 h of treatment (P < 0.05). When HC11 cells were cultured in medium containing lactogenic hormones prolactin and glucocorticoids, treatment of the cells with 0.1–25 µg/mL of LPS for 3 h and 24 h reduced β-casein gene (CSN2) expression by 40–80% (P < 0.05), but surprisingly increased αS1-casein (CSN1S1) expression by 20–226 fold (P < 0.05). Expression of α-lactalbumin gene (LALBA) was also increased at low concentrations of LPS (0.5 and 1 µg/mL) at 3 h, but decreased by 37–72% at all concentrations tested at 24 h (P < 0.05). In summary, our data demonstrated novel differential effects of LPS on expression of 3 major milk protein genes in MECs, suggesting potential functional differences among these proteins during mastitis. Especially, the dramatic rise of αS1-casein expression by LPS raised a possible immune function of this protein in the mammary gland.