Alternanthera brasiliana is a medicinal plant with several popular use and high production of betalains. The aim of the present research was to establish a protocol for callus induction and betalain biosynthesis in A. brasiliana species, as well as to investigate the effect of different qualities of light in increasing betalain in A. brasiliana callus cultures. In this way, the callus initiation and the improvement of secondary metabolites were induced using varying phytohormones concentrations and under the influence of red, white and blue lights. To start the experiments, the plants were cultivated for 30 days in Murashige and Skoog (MS) medium to grow and form internodes. These were inoculated in MS medium supplemented with different combinations of plant growth regulators to find the best combination for medium callus induction (MCI). After another 30 days, the explants were transferred to a medium for betacyanin induction (MBI) containing thidiazuron (TDZ) and α-naphthalene acetic acid (NAA) under blue, white, and red lights. The best medium for A. brasiliana callus induction with higher production of betalains, was the MS medium supplemented white indole-3-acetic acid (IAA) and 2,4dichlorophenoxyacetic acid (2,4-D), before inoculation on the MBI. The blue and white lights promoted callus pigmentation (betalains), whereas the red light was not effective at inducing pigmentation in the calli.