Mouse ARD1 (mARD1) has been reported to negatively regulate the hypoxia-inducible factor 1alpha (HIF-1alpha) protein by acetylating a lysine residue and enhancing HIF-1alpha ubiquitination and degradation. However, it was recently reported that human ARD1 (hARD1) does not affect HIF-1alpha stability. To further explore the activities of the two orthologs, three mouse (mARD1(198), mARD1(225), mARD1(235)) and two human (hARD1(131), hARD1(235)) variants were identified and characterized. Among these, mARD1(225) was previously reported as a novel negative regulator of HIF-1alpha. Amino acid sequence analysis showed that the C-terminal region (aa 158-225) of mARD1(225) completely differs from those of mouse and human ARD1(235), although all three proteins share a well-conserved N-acetyltransferase domain (aa 45-130). The effects of ARD1 variants were evaluated with respect to HIF-1alpha stability and acetylation activity. Interestingly, mARD1(225) strongly decreased the level of HIF-1alpha and increased the extent of acetylation, whereas mARD1(235) and hARD1(235) variants had a much weaker effect on HIF-1alpha stability and acetylation. These results suggest that ARD1 variants might have different effects on HIF-1alpha stability and acetylation, which may reflect diverse biological functions that remain to be determined.