Scheffersomyces stipitis shows a high capacity to ferment xylose, with a strong oxygen dependence to allow NAD+ regeneration. However, without oxygen regeneration of NADH occurs by other metabolic pathways like alcoholic fermentation. There are few reports about inhibitors of mitochondrial respiration and their effects on growth and fermentation. This work aimed to explore the effect of cytochrome bc1 complex inhibition by antimycin A (AA), on growth and fermentation of S. stipitis using glucose, xylose and arabinose as carbon sources, at three agitation levels (0, 125 and 250 rpm). It was possible to discriminate between respiratory and fermentative metabolism in these different conditions using xylose or arabinose. Despite the inhibition of mitochondrial respiration, the glycolytic flux was active because S. stipitis metabolized glucose or xylose to produce ATP; on 0.5 M glucose the cells yielded 17-33 g L-1 ethanol. However, more complex results were obtained on xylose, which depended upon agitation conditions where ethanol production without agitation increased up to 11 g L-1. Inhibition of respiratory chain in S. stipitis could therefore be a good strategy to improve ethanol yields.