The purposive manipulation of photoperiod condition that could delay or inhibit flowering in stevia (Stevia rebaudiana Bertoni) both under in vitro and ex vitro conditions was investigated. Under in vitro condition, stevia did not flower under either short-day or long-day condition; instead, leaf production was promoted. In contrast, ex vitro grown plants, whether potted or directly planted to the field, flowered under photoperiods shorter than the established critical daylength of 13 h for stevia and were inhibited from flowering at longer photoperiod of 15 h. Flowering was not prevented in stevia obtained from plants that have previously flowered even when exposed to long photoperiods. The steviol glycoside (SG) accumulation was lower in in vitro grown plants than those of plants maintained under ex vitro conditions; the stevioside content ranged from 0.83%−0.90%, while rebaudioside A amounted to only 0.26%−0.42%. Under ex vitro conditions, the average leaf stevioside content of tissue culture-derived stevia was 5.64%, while that of the flowers was 3.82%; rebaudioside A was 0.96% and 0.48% in these organs, respectively. The purposeful manipulation of photoperiod may be useful to stevia growing and SG accumulation both under in vitro and ex vitro production systems.