Aims: To optimize a medium for nicotine degradation by Ochrobactrum intermedium DN2 in presence of yeast extract, glucose and Tween 80 using response surface methodology (RSM). Methods and Results: In this study, the effects of yeast extract, glucose and Tween 80 on nicotine degradation were investigated in flasks using a novel nicotine-degrading bacterium, O. intermedium DN2. A full factorial central composite design was applied in the design of experiments and in the analysis of the experimental data. The results showed that the most significant variable influencing nicotine degradation was yeast extract, followed by glucose, and then Tween 80. Moreover these three factors interacted with each other and combined to produce positive effects on nicotine degradation. The experimental data also allowed the development of an empirical model (P < 0.0001) describing the inter-relationship between independent and dependent variables. By solving the regression equation, the optimal values of the variables were determined as: yeast extracts 0.094%, glucose 0.101% and Tween 80 0.080%. Using the medium obtained, about 1220 mg l(-1) of nicotine was degraded (95.55%) within 10 h at the specific biodegradation of 116.59 mg l(-1) h(-1) in 30-l bioreactor containing 25-l tobacco extract. Conclusions: An optimal medium of nicotine degradation by the strain DN2 was obtained. Significance and Impact of the Study: RSM proved to be reliable in developing the model, optimizing factors and analysing interaction effects. The results provide better understanding on the interactions between yeast extract, glucose and Tween 80 for nicotine biodegradation.