Ribonucleotide reductase (RNR) is an essential enzyme that catalyzes the rate-limiting step in the conversion of ribonucleotides (rNTP) to concomitant deoxyribonucleotides (dNTP) during de novo DNA synthesis [1, 2]. RNR is a heterodimeric tetramer consisting of α2β2 heterodimers, where α2 denote the larger R1 subunit which serves as the catalytic site for rNTP reduction and β2 denote the R2 subunit which serves a regulatory role in conjunction with the R1 catalytic counterpart and function in S-phase DNA replication. Mammals also have additional regulatory RNR protein p53R2 (encoded by RRM2B gene) that function during DNA repair, hypoxia, and mitochondrial DNA synthesis [3, 13]. The R1 and R2/p53R2 subunits together form the active functional enzyme and these subunits are endowed with reactive thiols which are susceptible to redox modifications. R1 subunit has catalytically active cysteine residues at the active site and a C-terminal swinging arm bearing Cys-Leu-Met-Cys sequence that behaves as shuttle dithiol/disulfide for electron transport during enzyme turn over and reduction cycles. R2/p53R2 subunit also bears tyrosyl free radical which allows them to participate in the free radical mechanism of reduction [4]. RNRs are evolutionarily conserved across all species and play an essential role in maintaining dNTP pools for the proper regulation of the rate of DNA synthesis to cell volume during cell division and repair. It is due to this fundamental role, RNRs are attractive targets for anti-cancer and anti-viral drugs. During rNTP reduction, the R1 active site cysteine forms a disulfide after each cycle which is reduced further by shuttle dithiols of the C-terminal swinging arm via accepting electrons from Thioredoxin (Trx) or glutathione (GSH)-glutaredoxin (Grx) systems. The disulfide formed in turn at the shuttle dithiol is further reduced by GSH-Grx or Trx systems [4, 5]. The mechanism of RNR mediated catalysis is largely aided by redox modification S-glutathionylation which depends upon the availability of GSH-Grx electron donor systems. S-glutathionylation is the covalent addition of glutathione to thiols of proteins leading to the formation of disulfide bonds. Avval et al. reported that Trx and GSH-Grx systems are hydrogen donors for RNR catalysis [6]. The C-terminal tail of the R1 subunit reportedly underwent S-glutathionylation which facilitated the RNR catalysis. Recently, we have reported that GSH-Grx system is a more efficient electron donor for RNR function with catalytic efficiency 4–6 fold higher than Trx, and GSH-Grx complex favors R1-p53R2 systems during DNA repair and replicative stress [7]. GSH formed adduct with the R1 subunit in a concentration-dependent manner presumably at a higher concentration (5–10 mM) with R1-R2 or R1-p53R2 complex during the catalysis and provided electrons to the shuttle dithiols for efficient reduction of active site thiols, which would otherwise not be reduced by redoxins themselves due to sterically narrow active site pocket of R1 [8, 4]. This novel mechanism allowed RNR to maintain its active site thiols in reduced state. This also facilitated RNR to maintain its activity in hypoxic and/or even in low Trx environment as occurs in the tumor microenvironment and prolonged DNA synthesis [7, 13]. GSH concentration however gets up-regulated in cancer cells due to metabolic reprogramming which has been well documented to support tumor progression and tumor resistance [9]. S-glutathionylation has also been reported to contribute to cancer progression and treatment resistance [10]. This can also provide a reasonable explanation why the GSH-Grx system is a more efficient electron donor for RNR disulfide reduction and sustain high RNR activity. However, no such data relating to R2 or mammalian p53R2 subunit S-glutathionylation has been made so far. Herein, we report for the first time S-glutathionylation of the R2 subunit of RNR in cell free assay system and we have also shown that Grx1 can efficiently deglutathionylate the S-glutathionylated R2 subunit (R2-SSG). Additionally, we also report S-glutathionylation of p53R2 subunit similar to R2 subunit.