SIRT5 is one of the seven mammalian sirtuins which are NAD+-dependent deacylases. In human beings, SIRT5 gene encodes for four SIRT5 protein isoforms, namely SIRT5iso1, SIRT5iso2, SIRT5iso3, and SIRT5iso4. Previous studies have focused mostly on SIRT5iso1. Characteristics regarding localization, activity and tissue distribution of the other three SIRT5 isoforms remain unclear. In the present study, we characterized these properties of these SIRT5 isoforms. We found that SIRT5iso1−3 were mitochondria-localized, while SIRT5iso4 localized mainly in cytoplasm. SIRT5iso2−4 had little deacylase activity comparing with SIRT5iso1. Although cDNAs of all SIRT5 isoforms were readily detected in multiply tissues according to EST database, proteins of SIRT5iso2−4 were seldom observed in human cell lines. Altogether, we dissected the four isoforms of human SIRT5 protein.