KAE609 [(1′R,3′S)-5,7′-dichloro-6′-fluoro-3′-methyl-2′,3′,4′,9′-tetrahydrospiro[indoline-3,1′-pyridol[3,4-b]indol]-2-one] is a potent, fast-acting, schizonticidal agent in clinical development for the treatment of malaria. This study investigated the absorption, distribution, metabolism, and excretion of KAE609 after oral administration of [14C]KAE609 in healthy subjects. After oral administration to human subjects, KAE609 was the major radioactive component (approximately 76% of the total radioactivity in plasma); M23 was the major circulating oxidative metabolite (approximately 12% of the total radioactivity in plasma). Several minor oxidative metabolites (M14, M16, M18, and M23.5B) were also identified, each accounting for approximately 3%–8% of the total radioactivity in plasma. KAE609 was well absorbed and extensively metabolized, such that KAE609 accounted for approximately 32% of the dose in feces. The elimination of KAE609 and metabolites was primarily mediated via biliary pathways. M23 was the major metabolite in feces. Subjects reported semen discoloration after dosing in prior studies; therefore, semen samples were collected once from each subject to further evaluate this clinical observation. Radioactivity excreted in semen was negligible, but the major component in semen was M23, supporting the rationale that this yellow-colored metabolite was the main source of semen discoloration. In this study, a new metabolite, M16, was identified in all biologic matrices albeit at low levels. All 19 recombinant human cytochrome P450 enzymes were capable of catalyzing the hydroxylation of M23 to form M16 even though the extent of turnover was very low. Thus, electrochemistry was used to generate a sufficient quantity of M16 for structural elucidation. Metabolic pathways of KAE609 in humans are summarized herein and M23 is the major metabolite in plasma and excreta.