The present study investigated growth properties and flowering response of seed-propagated strawberry (Fragaria ×ananassa) seedlings under artificial lighting with different photoperiods to support the development of a high-performance system for the indoor production of strawberry plug transplants. Seedlings of ‘Elan’ and ‘Yotsuboshi’ were grown for 38 days under sunlight in a greenhouse or under light-emitting diode (LED) illumination with photoperiods of 8/16, 12/12, 16/8, or 24/0 hours (light/dark) in growth chambers. The photosynthetic photon flux (PPF) in these photoperiods was maintained at 350, 230, 175, or 115 μmol·m−2·s−1, respectively, to provide the same daily light integral (DLI) of 10 mol·m−2·d−1. The average of DLI of sunlight was 9.9 mol·m−2·d−1. Seedling growth was greater with the 16- and 24-hour photoperiods than with sunlight even though all three treatments provided about the same DLI. Flower buds of the seedlings grown under longer photoperiods started significantly earlier after transplanting in ‘Elan’ but not in ‘Yotsuboshi’. Thus, strawberry transplant production under artificial lighting with an optimized photoperiod can provide high-quality transplants, although the effectiveness is cultivar-specific.