In bacterial cells, RNase HI usually degrades RNA in the RNA/DNA hybrids to prevent the accumulation of primers in replication and the formation of R-loops in transcription, thus maintaining genomic stability and normal life activities. The recognition of substrates by RNase HI mainly depends on DNA- and RNA-binding grooves, and the catalysis of substrates by RNase HI mainly depends on the DEDD motif and a histidine located in a flexible loop near the active site. Metal ions represented by Mg²⁺ play an important role in the catalytic process. The mode of action of RNase HI is determined by the type of ssDNA overhangs on RNA/DNA hybrids. In the presence of a 5′ ssDNA overhang or in the absence of any overhang on RNA/DNA hybrids, RNase HI functions as a non-sequence-specific endonuclease to degrade RNA randomly. In the presence of a 3′ ssDNA overhang on RNA/DNA hybrids, RNase HI relies on 5′-exonuclease activity for the successive degradation of RNA. RNase HI, Rep, DinG, and UvrD are recruited near the replication forks by interacting with the six residues of the C-terminal tail of single-stranded DNA-binding protein (SSB), and may resolve replication-transcription conflicts in a cooperative manner. The deletion of RNase HI or the decrease in RNase HI activity will cause a series of harmful events such as DNA structural instability, gene mutation, transcriptional machinery backtracking, and replication incoordination. RNase HI has shown great application prospects in antisense technology, R-loop detection, and targeted therapy combined with antibiotics. The cooperative mechanism of primer degradation by RNase HI and other enzymes is also worth studying in the future.