Elements not usually included in culture medium formulations, such as selenium (Se), may have beneficial effects on micropropagated plants. We evaluated the effects of Se on the physiological and anatomical responses of Alcantarea imperialis during in vitro culture. Plants were cultured in a medium containing a gradient of Se concentrations (0, 4, 8, 16, or 32 µM Se). After 56 d, the growth traits, chlorophyll a fluorescence, and root and leaf anatomy were analyzed. The fresh mass declined at the highest Se concentration. Higher Se concentrations induced bigger stomata, while the stomatal density decreased. Plants cultured with Se had improved PSII and PSI electron transport. This led to higher values of the total performance index. Thus, Se-induced plants showed a higher electron transport dynamics and energy conservation from water to PSI and developed anatomical traits that can favor tolerance to water deficit.