To characterize uracil-DNA glycosylase from acidophilic and thermophilic Sulfolobus acidocaldarius.We cloned udgIV and udgV genes from S. acidocaldarius, expressed the two recombinant UDG proteins in E. coli species BL21 (DE3) Rosetta-pLysS, purified the recombinant UDGs and characterized the removal of dU by UDGs.We successfully expressed two S. acidocaldarius UDGs and found both UDGs having the activity of dU removal. In comparison to UDGV, UDGIV was more efficient in dU removal, with a 750-foldactivity.In comparison to UDGV, UDGIV from S. acidocaldarius was a more efficient enzyme responsible for the removal of dU from DNA in vitro.