Aeropyrum pernix contains one homolog of ribonuclease H (RNase H), A. pernix RNase HII (Ape-RNase HII). Activity characterization showed that Ape-RNase HII exhibited the highest activity in the presence of 5 mM Mn2+, 1 mM Co2+, or 10 mM Mg2+, respectively; however, its cleavage efficiencies at different cleavage sites for Mn2+ and Mg2+ were different. Ape-RNase HII cleaved 12-bp RNA/DNA substrates at multiple sites and the optimum pH value was 11.0. Moreover, 16-bp DNA-r4-DNA/DNA and 13-bp DNA-r1-DNA/DNA chimeric substrates were cleaved at DNA–RNA junction. Ape-RNase HII was thermostable and the stabilization was enhanced with increased salt concentration. This work is believed to be the first in vitro functional study of Ape-RNase HII and the results should contribute to the analysis of RNase H of other archaeal species.