Abstract: Because of long adaptation to the extreme environment of high pressure, low temperature, darkness and nutrientpoor waters, deep-sea microorganisms are bound to develop a corresponding molecular basis. In recent years, the mechanism of extreme environmental adaptability of deepsea microorganisms is a frontier topic in the study of deepsea microorganism. With atmospheric and room temperature plasma (ARTP) mutagenesis of a cold and pressure tolerant deep-sea actinomycetesMicrobacterium sediminis YLB-01, the mutant 9-3E5 which was sensitive to low temperature and high pressure was successfully obtained. The genome comparative analysis of the mutant 9-3E5 and the wild-type strain showed that the SNP, accounting for 77.45% of the total, and InDels were evenly distributed on the whole genome of mutant 9-3E5 and 838 SNP sites were located in the coding region and were nonsynonymous mutations. There were 11 InDels of frameshift mutation located in the coding region. The mutant 9-3E5 has 11 SV sequences and length 300-400 bp accounting for 81.82% of the total. According to the standard of OR > 2, p < 0.05, the mutant genes were enriched into 3 functional sets. Eleven SNP mutation sites were found in 4 cell division genes of FtsI, FtwW, FtsX and FtsZ. Ten SNP mutation sites were found in 6 genes related to DNA repair and synthesis. Three SNP mutation sites were found in 2 genes related to intracellular transport and energy synthesis regulation. We speculate that the changes of the tolerance of mutant 9-3E5 to low temperature and high pressure compared with wild strain YLB-01 may be due to the changes in three functional genes of cell division, repair and synthesis of DNA damage, and intracellular transport regulates energy synthesis. It is suggested that M. sediminis YLB-01 adjust these intracellular physiological and biochemical processes to cope with external low temperature and high pressure environment.