Enriching food with vitamin C is a process that challenges food engineers. To prevent the degradation of this vitamin, a microencapsulation can be used in Saccharomyces cerevisiae cells. Previous works have shown that applying a pulsed electric field can increase the efficiency of the accumulation of minerals in yeast. The aim of this study was to optimize PEF parameters in order to increase the accumulation of vitamin C in yeast cells, to evaluate the effect of electroporation on biomass and yeast viability, and to assess the effect of storage conditions on the vitamin C content and its antioxidant activity. The most effective accumulation of vitamin C in cells (approx. 1.3 mg/g dry mass) was achieved when a 20-h yeast culture was treated with PEF at a concentration of 5 mg/mL vitamin C in the medium. The optimal PEF parameters were: voltage of 1000 V, pulse width of 10 μs, treatment time of 20 min, and number of pulses, 1200. The process conditions did not affect significantly biomass production nor cell viability. Yeast cells with vitamin C were stored for 7, 14, and 28 days at 20 ◦C (after prior freeze-drying), 4 ◦C, and −22 ◦C. The lowest decrease in vitamin C content was observed for the freeze-dried yeast stored at 20 ◦C.