The objective of this study was to estimate the in vitro activity of egg yolk immunoglobulin (IgY) against mastitis-causing Escherichia coli. Specific IgY was produced by hens immunized with formaldehyde killed E. coli O111 in long-standing immunization response (titer > or =6400 for 100 days) and was isolated from yolks with a purity of 86% by water dilution, salt precipitations and ultrafiltration. Enzyme-linked immunosorbent assay (ELISA) indicated the produced IgY specifically targeted E. coli O111 and five other E. coli strains which were isolated from mastitic cows. The growth inhibition activity of the specific IgY to bacteria was dose-dependent with an effective concentration of 20mg purified IgY per milliliter. The phagocytic activity of E. coli either by milk macrophages (MPhi) or by polymorphonuclear neutrophil leukocytes (PMN) in the presence of specific IgY was significantly higher than that with nonspecific IgY or without IgY (p<0.05), suggesting that it enhanced phagocytic activity. The current work suggests that this specific IgY has potential as a therapeutic treatment for mastitis in dairy cows.