Aims: The aim of this study is to clarify the effect of Tween 80 on bile tolerance of lactococci. Methods and Results: Four out of the six strains of lactococci could grow in broth containing 0.3% bile in the presence of 1% Tween 80, but grew slightly or not at all in the absence of Tween 80. Growing with Tween 80 altered the fatty acid composition of all three strains tested, but it is not clear which fatty acid influences bile tolerance. Material that absorbed light at 260 nm leaked from the cells tested with bile, but the leakage was decreased by addition of 1% Tween 80. Coincidentally, the decrease in the cell count by exposure to bile was suppressed by addition of Tween 80. Conclusions: Tween 80 enhances bile tolerance of some strains of lactococci. Significance and Impact of the Study: :It is clarified that Tween 80 in the broth contributes to the bile tolerance of lactococci by reducing the cellular leakage caused by bile.