Mutants lacking a thylakoid membrane-bound metalloprotease, FtsH, are known to cause leaf variegation in Arabidopsis. However, the effect of reduced FtsH levels on leaf variegation has scarcely been examined in other plants. In this study, we performed RNA interference (RNAi) by which FtsH expression was suppressed in tobacco. The resulting FtsH knock-down tobacco plants showed variegation in their leaves, and a negative correlation between the degree of variegation and the level of FtsH, which supported earlier observations in Arabidopsis. A decrease of NtFtsH2 as well as NtFtsH1 suggested that these are the two major isoforms comprising the FtsH complex in tobacco chloroplasts. The RNAi tobacco lines also showed photoinhibition-vulnerable phenotypes, as evidenced by high-light-sensitive PSII activity and retarded degradation of D1 protein. Interestingly, the formation of variegated sectors during leaf development appeared to differ between Arabidopsis and tobacco. In contrast to the formation of variegation in Arabidopsis, the yellow sectors in FtsH RNAi tobacco emerged from green leaves at a late stage of leaf development. A series of cytological observations implied that thylakoid membranes were dismantled after development had already occurred. Late formation of variegation in FtsH RNAi tobacco suggested that the heteromeric FtsH complex is important for maintaining thylakoid membranes.