Lactational mastitis is a common inflammatory breast disease in postpartum women. Although bacterial pathogens are generally considered responsible for mastitis, there are inconsistencies between disease severity and bacterial load, and scarce evidence that antibiotic treatment is effective in treating or preventing mastitis in women. The inflammatory pathways activated in the host are likely to affect disease progression and resolution. We developed a new mouse model of the disease to investigate the role of toll-like receptor 4 (TLR4) signalling in mastitis. Lipopolysaccharide in matrigel (10ug/10uL) was administered into the duct of lactating Tlr4 null mutant and wildtype control mice to induce a localised area of inflammation in the mammary gland. Serum cytokine concentration and histology of the mammary gland were analysed over the following 7 days. In control mice, mastitis induction caused a marked influx of RB6-positive neutrophils and F4/80-positive macrophages into the affected mammary gland, and increased serum CXCL1 and IL10 concentration. In Tlr4-/- mice, a 100% increase in neutrophils and 83% increase in macrophages was evident 8 hours and 1 day post mastitis induction respectively compared to control mice (p<0.05). Serum CXCL1 and IL10 concentration 4 hours following mastitis induction was attenuated 14-fold and 7-fold in Tlr4-/- mice respectively (p<0.05). In both genotypes, the localised area of inflammation had resolved after 7 days, however there was evidence of functional lactation in the affected mammary gland in only 40% (4/10) of control mice compared to 82% (9/11) of Tlr4-/- mice. Non-functional glands appeared to have undergone involution. This study demonstrates that the specific inflammatory pathways activated in the host are critically important for mastitis disease progression and resolution, and that the absence of functional TLR4 protects the mammary gland and lactation in mastitis.