Preliminary findings indicate that PprI is a regulatory protein that stimulates transcription and translation of recA and other DNA repair genes in response to DNA damage in the extreme radioresistance bacterium Deinococcus radiodurans. To define the repertoire of proteins regulated by PprI and investigate the in vivo regulatory mechanism of PprI in response to gamma radiation, we performed comparative proteomic analyses on wild type (R1) and a pprI knockout strain (YR1) under conditions of ionizing irradiation. Results of 2-dimensional electrophoresis and MALDI-TOF MS or MALDI-TOF/TOF MS indicated that in response to low dose gamma ray exposure, 31 proteins were significantly up-regulated in the presence of PprI. Among them, RecA and PprA are well known for their roles in DNA replication and repair. Others are involved in six different pathways, including stress response, energy metabolism, transcriptional regulation, signal transduction, protein turn-over and chaperoning. The last group consists of many proteins with uncharacterized functions. Expression of an additional four proteins, most of which act in metabolic pathways, were down-regulated in irradiated R1. Additionally, phosphorylation of two proteins was under the control of PprI in response to irradiation. The different functional roles of representative PprI-regulated genes in extreme radioresistance were validated by gene knockout analysis. These results suggest a role, either directly or indirectly, for PprI as a general switch to efficiently enhance the DNA repair capability and extreme radioresistance of D. radiodurans via regulation of a series of pathways.