BACKGROUND AND PURPOSE Leukotrienes (LTs) are pro‐inflammatory mediators produced by 5‐lipoxygenase (5‐LO). Currently available 5‐LO inhibitors either lack efficacy or are toxic and novel approaches are required to establish a successful anti‐LT therapy. Here we provide a detailed evaluation of the effectiveness of the plant‐derived alkaloid tryptanthrin as an inhibitor of LT biosynthesis. EXPERIMENTAL APPROACH We analysed LT formation and performed mechanistic studies in human neutrophils stimulated with pathophysiologically relevant stimuli (LPS and formyl peptide), as well as in cell‐free assays (neutrophil homogenates or recombinant human 5‐LO) and in human whole blood. The in vivo effectiveness of tryptanthrin was evaluated in the rat model of carrageenan‐induced pleurisy. KEY RESULTS Tryptanthrin potently reduced LT‐formation in human neutrophils (IC50= 0.6 µM). However, tryptanthrin is not a redox‐active compound and did not directly interfere with 5‐LO activity in cell‐free assays. Similarly, tryptanthrin did not inhibit the release of arachidonic acid, the activation of MAPKs, or the increase in Ca2+i, but it modified the subcellular localization of 5‐LO. Moreover, tryptanthrin potently suppressed LT formation in human whole blood (IC50= 10 µM) and reduced LTB4 levels in the rat pleurisy model after a single oral dose of 10 mg·kg−1. CONCLUSIONS AND IMPLICATIONS Our data reveal that tryptanthrin is a potent natural inhibitor of cellular LT biosynthesis with proven efficacy in whole blood and is effective in vivo after oral administration. Its unique pharmacological profile supports further analysis to exploit its pharmacological potential.