Heme oxygenase (HO) is a rate-limiting step of heme degradation, which catalyzes the conversion of heme into biliverdin, iron, and CO. HO has been characterized in micro-organisms, insects, plants, and mammals. The mammalian enzyme participates in adaptive and protective responses to oxidative stress and various inflammatory stimuli. The present study reports the use of RNA-interference (RNAi) to suppress HO in the multicellular eukaryote Drosophila. Eye imaginal disc-specific suppression of the Drosophila HO homolog (dHO) conferred serious abnormal eye morphology in adults. Deficiency of the dHO protein resulted in increased levels of iron and heme in larvae. The accumulation of iron was also observed in the compound eyes of dHO-knockdown adult flies. In parallel with the decrease of dHO, the expression of delta-aminolevulinic acid synthase, the first enzyme of the heme-biosynthetic pathway, in larvae was decreased markedly, suggesting that heme biosynthesis was totally suppressed by dHO-deficiency. The activation of caspase-3 occurred in eye imaginal discs of dHO-knockdown flies, indicating the occurrence of apoptosis in the discs. On the other hand, the overexpression of dHO resulted in a weak but significant rough eye phenotype in adults. Taken together, considering that dHO is not a stress-inducible protein, the expression of dHO can be tightly regulated at developmental stages and the relevant expression is necessary for the normal development of tissues in Drosophila.