The influences of Tween 80 on the pullulan production, cell growth, glucosyltransferase activity and fatty acid composition of the cells were studied. The addition of Tween 80 to the culture medium significantly enhanced the pullulan production, but had no impact to biomass accumulation. Glucosyltransferase activity involved in pullulan synthesis was remarkable promoted with an increase of Tween 80 content. The ratio of unsaturated/saturated fatty acids contained in the cells was significantly higher in 0.5% Tween 80 in comparison with the absence of Tween 80. Results indicated that the increase of pullulan yield brought by Tween 80 was highly correlated with glucosyltransferase activity and fatty acid composition. Tween 80 was not degraded to serve as extra carbon source.