Plants developed several strategies to cope with ultraviolet radiation (UV) exposure, including accumulation of specialized metabolites, with a recognized major role played by flavonoids. The unique alkaloid brachycerine of Psychotria brachyceras (Rubiaceae) is induced by leaf exposure to high UV intensity and is a potent antioxidant, so it is likely to participate as a protecting factor against this radiation. The capacity of brachycerine as a putative agent in acute UV protection was tested by comparison of P. brachyceras with P. carthagenensis, a closely related and co-ocurring species devoid of alkaloids, in UV-B exposure experiments. Leaves of P. brachyceras were very tolerant to high UV doses when compared with P. carthagenensis, lacking chlorophyll loss and membrane lipid peroxidation. Overall, well known leaf defenses against UV-B, such as flavonoids and cuticular waxes, were not markedly different between Psychotria species. The alkaloid was found both in the epidermis and mesophyll in leaves of P. brachyceras. Brachycerine is an effective in vitro superoxide scavenger and, when applied to the surface of P. carthagenensis leaves, significantly increased UV-B tolerance, improving chlorophyll concentration and several photosynthetic parameters under this treatment. Profiles of brachycerine extracted from alkaloid-treated P. carthagenensis leaves showed partial degradation after UV exposure, suggesting alkaloid involvement in chemical quenching of reactive oxygen species. In addition to flavonoids, the alkaloid brachycerine may improve tolerance to acute UV-B, representing a chemical tool to protect plants against this harmful condition. © 2020 Elsevier B.V.