To explore the possible regulatory role of chlorophyllase (Chlase) in chlorophyll (Chl) degradation during leaf senescence, RNAi Arabidopsis (Arabidopsis thaliana) plants were constructed to repress the expressions of AtCLH1 and/or AtCLH2. Transcript levels of AtCLH1 and/or AtCLH2 were dramatically lowered and Chlase activity was correspondingly inhibited, but the Chl degradation kinetics was not affected in the RNAi plants. Results of further analysis indicated that the Chl a/b ratio decreased in AtCLH1 RNAi lines, in comparison with the increasing Chl a/b ratio in the wide type during leaf senescence. In addition, an induced Chlase activity was consistently detected at the initial stage of senescence in all the plants examined. In contrast, transcript levels of both AtCLH1 and AtCLH2 decreased dramatically upon the initiation of senescence in both the wide-type and the RNAi plants. Interestingly, compared with the wide type, lower but still significant transcript levels of the RNAi targeted Chlase gene(s) were sustained during the whole period of dark incubation in all the three RNAi lines examined, indicating the functioning of some compensatively regulating mechanism. Based on these results, along with related reports, we conclude that Chlase might be required at the initial stage of leaf senescence, quite likely playing a role in converting Chl b to a.