8‐Nitroguanosine 3′,5′‐cyclic monophosphate (8‐nitro‐cGMP) is a nitric oxide metabolite and an important second messenger. 8‐Nitro‐cGMP reacts with sulfhydryl groups forming a novel posttranslational modification, namely, S‐guanylation. In this work, we found, by using a quantitative competition enzyme‐linked immunosorbent assay procedure, that S‐guanylated human serum albumin (S‐cGMP‐HSA) is a component of normal plasma, and that hemodialysis patients decrease its concentration, on an average, from 68 to 34 nM. End‐stage renal disease is often accompanied by septicemia, and we found that S‐cGMP‐HSA possesses an in vitro antibacterial effect with half maximal inhibitory concentration of approximately 2 μM against Escherichia coli American Type Culture Collection. Our findings indicate that S‐cGMP‐HSA can be regarded as an endogenous antibacterial agent in healthy conditions and as a useful new class of antibacterial agents with a circulation time sufficient for in vivo biological activity. The clinical development of S‐cGMP‐HSA as a safe and strong antibacterial agent arisen from endogenous posttranslational modification would be expected. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:3222–3229, 2012