The concentration of lactoferrin (LTF) in milk varies during lactation, rising sharply during involution. We proposed that LTF might have a regulatory role in involution and investigated its effects in vitro on the viability of bovine mammary epithelial cells (BMEC) and on casein expression in bovine mammospheres. Mammospheres capable of milk protein expression were formed by culturing primary BMEC on extracellular matrix in the presence of lactogenic hormones. Exogenously added LTF decreased ÃŸ-casein and '-casein mRNA expression in mammospheres while transfection of a short interfering RNA (siRNA) to suppress LTF expression resulted in increased casein mRNA expression. We believe that LTF exerts its effect on casein gene expression by up-regulating interleukin-1ÃŸ (IL-1ÃŸ) as IL-IÃŸ gene expression was elevated in mammospheres treated with LTF. LTF also decreased viability of BMEC grown as monolayers and as mammospheres. Interestingly, LTF was only effective in reducing casein mRNA expression and viability in mammospheres when added at concentrations found during early involution but was inactive when used at concentrations found in milk. We suggest that LTF has a regulatory role during early involution, decreasing casein expression and reducing BMEC viability.