Hyptis marrubioides Epling, Lamiaceae, a species from Brazilian Cerrado, has been used against gastrointestinal infections, skin infections, pain, and cramps. Herein, H. marrubioides seedlings were cultured in vitro under different wavelengths (white, blue, green, red, and yellow) with 50μmolm−2s−1 irradiance and a 16-h photoperiod. After 20 and 30 days of cultivation, shoot length, leaf number, fresh mass, and dry mass were evaluated. The flavonoid rutin content was determined by the HPLC-DAD method. The shoots were longer in plants cultivated under yellow (16.603±0.790cm, 1.8-fold), red (15.465±0.461cm, 1.7-fold), and green (14.677±0.737cm, 1.6-fold) lights than in control plants exposed to white light (9.203±0.388cm). The number of leaves increased in plants exposed to red (23.425±1.138, 1.1-fold) and green (22.725±1.814, 1.1-fold) lights, compared to control plants (20.133±0.827). Fresh (0.665±0.048g, 1.2-fold) and dry (0.066±0.005g, 1.3-fold) mass of seedlings were the highest in seedlings grown under red light, compared to seedlings grown under white light (0.553±0.048 and 0.028±0.004, respectively). However, rutin production was higher under white (0.308mgg−1 of dry weight) and blue lights (0.298mgg−1 of dry weight). Thus, red light induces plant growth and increases leaf number and dry weight in in vitro-cultivated H. marrubioides, whereas blue and white lights promote the greatest rutin accumulation.