Objective DNA end resection is a common mechanism for the formation of 3"-ssDNA tails in homologous recombination (HR) and is mainly mediated by 5"-3" exonuclease. However, whether DNA end configurations directly regulate 5"-3" exonuclease activity remains unclear. In this study, we explored the regulation and mechanisms of DNA end configurations on RecJ, the only 5"-3" exonuclease of RecF recombination pathway in Escherichia coli.Methods To investigate the regulation of DNA 3"-end configurations on RecJ exonuclease, single-stranded DNAs (ssDNAs) containing different lengths of 3"-ssDNA overhangs (9 nt and 18 nt) and 3"-end modifications (phosphorylation and phosphorothioation) were used for exonuclease assays in the presence of Mg²⁺. To elucidate the mechanisms, RecJ was incubated with substrates containing different 3"-end configurations in the absence of Mg²⁺ and analyzed by electrophoretic mobility shift assays (EMSA). Furthermore, the coordination of DNA 3" end configurations and two other RecJ regulatory factors, DNA 5"-end phosphorylation and single-stranded DNA binding protein (SSB), were determined by exonuclease assays and EMSA on substrates with different 3"-end configurations respectively.Results DNA 3"-end configurations inhibited the RecJ exonuclease activity, including DNA 3"-overhang length and 3"-end modifications (phosphorylation and phosphorothioation). 3"-End phosphorylation and phosphorothioation of DNA reshaped the RecJ-DNA binding patterns to inhibit RecJ exonuclease activity. DNA 5"-end phosphorylation overcame RecJ inhibition of 3"-end modifications and remodeled the RecJ-DNA binding patterns. In addition, SSB partially overcame the 3"-end modifications mediated inhibition by enhancing RecJ-DNA binding.Conclusion The RecJ exonuclease activity was regulated and orchestrated by the DNA configurations of 3" and 5" ends.