Thymosin alpha 1 (Tα1) is an important immunomodulating agent with various clinical applications. The natural form of Tα1 is -acetylated， which was supposed to be related to stability of the hormone. In this study， fusion protein Tα1-HSA was constructed and expressed in . RimJ， a -acetyltransferase from ， was also overexpressed and purified to homogeneity. acetylation of Tα1-HSA in the presence of RimJ and acetyl coenzyme A resulted in -acetyl Tα1-HSA. The -acetylation was determined by LC-MS/MS. Kinetic assay indicated that RimJ had a higher affinity to desacetyl Tα1 than to Tα1-HSA. Bioactivity assay revealed fully retained activity of Tα1 when the hormone was connected to the N-terminus of the fusion protein， while the activity was compromised in our previously constructed HSA-Tα1. With fully retained activity and N-terminal acetylation， -acetyl Tα1-HSA was expected to be a more promising pharmaceutical agent than Tα1.