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Summary The ATP-dependent ClpQY system is a prokaryotic proteasome-like multi-subunit machinery localized in the mitochondrion of malaria parasite. The ClpQY machinery consists of ClpQ threonine pro- tease and ClpY ATPase. In the present study, we have assessed cellular effects of transient interference of PfClpQ protease activity in Plas- modium falciparum using a trans-dominant nega- tive approach combined with FKBP degradation domain system. A proteolytically inactive mutant PfClpQ protein (PfClpQ(mut)) fused with FKBP degradation domain was expressed in parasites, which gets stabilized by Shield1 drug treatment. We show that the inactive PfClpQ(mut) interacts with wild-type PfClpQ and associates within multi- subunit complex in the parasite. Stabilization of the PfClpQ(mut) and its association in the protease machinery caused dominant negative effect in the transgenic parasites, which disrupted the growth cycle of asexual blood stage parasites. The mito- chondria in these parasites showed abnormal mor- phology, these mitochondria were not able to grow and divide in the parasite. We further show that the dominant negative effect of PfClpQ(mut) disrupted transcription of mitochondrial genome encoded genes, which in turn blocked normal development and functioning of the mitochondria.