ABSTRACT Encephalitozoon cuniculi is a microsporidium responsible for systemic illness in mammals. In the course of developing leads to new therapy for microsporidiosis, we found that a bis(phenylbenzyl)3-7-3 analog of spermine, 1,15-bis{ N -[ o -(phenyl)benzylamino}-4,12-diazapentadecane (BW-1), was a substrate for an E. cuniculi amine oxidase activity. The primary natural substrate for this oxidase activity was N ′-acetylspermine, but BW-1 had activity comparable to that of the substrate. As the sole substrate, BW-1 gave linear reaction rates over 15 min and K _m of 2 μM. In the presence of N ′-acetylspermine, BW-1 acted as a competitive inhibitor of oxidase activity and may be a subversive substrate, resulting in increased peroxide production. By use of ¹³ C-labeled BW-1 as a substrate and nuclear magnetic resonance analysis, two products were determined to be oxidative metabolites, a hydrated aldehyde or dicarboxylate and 2(phenyl)benzylamine. These products were detected after exposure of ¹³ C-labeled BW-1 to E. cuniculi preemergent spore preparations and to uninfected host cells. In previous studies, BW-1 was curative in a rodent model of infection with E. cuniculi . The results in this study demonstrate competitive inhibition of oxidase activity by BW-1 and support further studies of this oxidase activity by the parasite and host.