Abstract Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, Grx3), all containing the classical dithiol active site CPYC. In this work, we are focus at Grx4, a newly found glutaredoxin in E. coli, encoded by gene grxD. The protein consists of 115 amino acids (ca. 12.7 kDa), has a potential monothiol (CGFS) active site. The grxD gene was reported as an essential gene in Escherichia coli. In this work, we tried different strategy to make a disruption mutant and suspect that the gene is essential for E. coli; however, only a two-copy “knock out” mutant was obtained. In the growth test, the two-copy “knock out” mutant grew slowly, but after obtaining another wild-type grxD copy, the growth condition was compensated. In the protein purification, a Grx4-6xHis recombination protein was constructed, and then purified with an affinity resin. As examined with SDS-PAGE, the protein size is 12.7kDa, matching the theoretical size. At the last, Grx4 may have an interaction with ClpY protein, a part of protease ClpYQ in E. coli. In this work, the yeast two-hybrid system was performed on the ClpY mutants and Grx4 protein, thus it is likely the result that Grx4 would be a substrate of ClpY protease in vivo, and thus implying that ClpY might recognize and bind the substrate with the I domain.