Trypanosoma brucei is the causing agent of African trypanosomiasis. These parasites possess a unique thiol redox system required for DNA synthesis and defense against oxidative stress. It includes trypanothione and trypanothione reductase (TryR) instead of the thioredoxin and glutaredoxin systems of mammalian hosts. Here, we show that the benzisothiazolone compound ebsulfur (EbS), a sulfur analogue of ebselen, is a potent inhibitor of T. brucei growth with a favorable selectivity index over mammalian cells. EbS inhibited the TryR activity and decreased non-protein thiol levels in cultured parasites. The inhibition of TryR by EbS was irreversible and NADPH-dependent. EbS formed a complex with TryR and caused oxidation and inactivation of the enzyme. EbS was more toxic for T. brucei than for Trypanosoma cruzi, probably due to lower levels of TryR and trypanothione in T. brucei. Furthermore, inhibition of TryR produced high intracellular reactive oxygen species. Hydrogen peroxide, known to be constitutively high in T. brucei, enhanced the EbS inhibition of TryR. The elevation of reactive oxygen species production in parasites caused by EbS induced a programmed cell death. Soluble EbS analogues were synthesized and cured T. brucei brucei infection in mice when used together with nifurtimox. Altogether, EbS and EbS analogues disrupt the trypanothione system, hampering the defense against oxidative stress. Thus, EbS is a promising lead for development of drugs against African trypanosomiasis.