Abstract The effects of important process parameters on the production of the novel prebiotic trisaccharide neokestose from sucrose by Xanthophyllomyces dendrorhous cells suspended in buffer were investigated. Recycling of cells reduced the level and rate of neokestose production while cells harvested in the exponential growth phase produced neokestose more efficiently than stationary phase cells. A further investigation into neokestose production was carried out using a fractional factorial statistical design. The neokestose produced was hydrolyzed following cessation of sucrose conversion at 31 g l −1 initial substrate concentration but not at 110 g l −1 initial sucrose. Statistical analysis of the data showed that sucrose concentration had a large positive effect on the maximum concentration of neokestose produced, the maximum specific rate of neokestose production and the maximum yield coefficient of neokestose. Surprisingly, increased cell concentration decreased the maximum specific rate of product formation. Strong interactions involving pH and temperature were identified. Curvature plots indicated that the optima for the maximum specific rate of neokestose production and for maximum yield coefficient were within the experimental range, whereas the optimum conditions for the maximum concentration of neokestose were outside this range. This is the first report on the production of a prebiotic oligosaccharide of the 6 G series in high yields.