The effect of Tween 80 on the fermentative production of polymalic acid (PMA) and pullulan using Aureobasidium pullulans CCTCC M2012223 was investigated. Tween 80 is beneficial for the biosynthesis of PMA and pullulan, and can regulate the ratio of PMA to pullulan in a dose-dependent manner. After adding 0.05 % Tween 80 to the media, the maximal PMA and pullulan production was 46.45 and 28.8 g/L at 60 h in a 5 L fermenter, with an increase of 75.08 and 27.21 % when compared to the control. Tween 80 could regulate and enhance oxygen uptake rate and carbon dioxide evolution rate in the early phase of fermentation, and change the cell morphology. The transcription levels of mitochondrial dicarboxylate transporter and transmembrane transporter were also dramatically upregulated. The present work will be helpful in deeply understanding the mechanism of Tween 80 on the effect of PMA and pullulan production.