We report the study of the dynamics of substrate utilization by the genetic modified strain Yarrowia lipolytica H222-S4(p67ICL1) T5. In contrast to its wild-type equivalent, this recombinant strain is able to excrete the sucrose cleaving enzyme invertase. Both the sucrose degradation rate and the glucose and fructose consumption rate have been investigated. In all experiments, satisfied amounts of invertase were produced so that all sucrose was cleaved into its monomers. While glucose and fructose as sole carbon sources were consumed with the same uptake rate, a clear preference for glucose uptake was detected in cultivations with sucrose as sole carbon source or mixed substrates when compared with fructose. Nevertheless, no real diauxie could be observed because of partly simultaneous consumption of both monosaccharides. Fructose being present in the cultivation medium at the beginning of the fermentation led to the retardation of glucose uptake. This effect was observed for various fructose starting concentrations in the range of 5–85 g/l.