DNA double-strand breaks (DSBs) are one of the most lethal forms of DNA damage that is not efficiently repaired in prokaryotes. Certain microorganisms can handle chromosomal DSBs using the error-prone non-homologous end joining (NHEJ) system and ultimately cause genome mutagenesis. Here, we demonstrated that Enterobacteria phage T4 DNA ligase alone is capable of mediating in vivo chromosome DSBs repair in Escherichia coli. The ligation efficiency of DSBs with T4 DNA ligase is one order of magnitude higher than the NHEJ system from Mycobacterium tuberculosis. This process introduces chromosome DNA excision with different sizes, which can be manipulated by regulating the activity of host-exonuclease RecBCD. The DNA deletion length reduced either by inactivating recB or expressing the RecBCD inhibitor Gam protein from λ phage. Furthermore, we also found single nucleotide substitutions at the DNA junction, suggesting that T4 DNA ligase, as a single component non-homologous end joining system, has great potential in genome mutagenesis, genome reduction and genome editing. Keywords: T4 DNA ligase, DNA double-strand breaks, Non-homologous end joining, CRISPR-Cas9