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Pulsed light is a rapid technology to convert ergosterol to vitamin D2 in mushrooms. Five Pleurotus mushrooms were used to study vitamin D2 generation using pulsed light, and the pulsed distances and pulses were also evaluated. In the different genera of Pleurotus mushrooms, P. ferulae produced the highest vitamin D2 content. The short pulsed distance (2.5 cm) and long pulsed time treatment (120 pulses) cause the reduction in the vitamin D2 content and discoloration in P. ferulae powder. Moreover, P. ferulae treated for 60 pulses at 5 cm was the optimal condition for vitamin D2 conversion (65.4 μg/g DW). The losses of vitamin D2 contents after 60 days of storage at 4 and 25C were 28.9 and 37.7%, respectively. Overall, irradiated P. ferulae would be a good source of vitamin D2 and pulsed light can be used to prepare mushrooms with high vitamin D2 content as a nutrient supplement. PRACTICAL APPLICATIONSPulsed light is showed to be a rapid technology for vitamin D2 conversion in mushrooms. This technology enhances the vitamin D2 content not only in fresh Pleurotus mushrooms but also in powdered Pleurotus mushrooms. Pulsed light irradiated mushroom powder is a good source of vitamin D2 and considered a nutrient supplement.