N (alpha)-Acetylation is one of the most common protein modifications in eukaryotes but a rare event in prokaryotes. Some endogenously N (alpha)-acetylated proteins in eukaryotes are frequently reported not to be acetylated or only very partially when expressed in recombinant Escherichia coli. Thymosin alpha 1 (T alpha 1), an N (alpha)-acetylated peptide of 28 amino acids, displays a powerful general immunostimulating activity. Here, we revealed that a fusion protein of thymosin alpha 1 and L12 is partly N (alpha)-acetylated in E. coli. Through deletion of some N (alpha)-acetyltransferases by Red recombination, we found that, when rimJ is disrupted, the fusion protein is completely unacetylated. The relationship of rimJ and N (alpha)-acetylation of T alpha 1 was further investigated by gene rescue and in vitro modification. Our results demonstrate that N (alpha)-acetylation of recombinant T alpha 1-fused protein in E. coli is catalyzed by RimJ and that fully acetylated T alpha 1 can be obtained by co-expressing with RimJ. This is the first description that an ectopic protein acetylation in bacterial expression systems is catalyzed by RimJ, a known prokaryotic N (alpha)-acetyltransferase.