HBx, a small regulatory protein of hepatitis B virus, plays an important role in stimulating viral genome replication. HBx was shown to be associated with diverse subcellular locations, such as the nucleus, cytoplasm and mitochondria. Some studies have linked the stimulation of genome replication by HBx to its cytoplasmic function, while other reports have attributed this function to its nuclear component. To clarify this discrepancy, we measured viral genome replication by complementing an HBx-null replicon in two different ways: by (i) co-transfecting with an increasing amount of HBx expression plasmid and (ii) co-transfecting with re-targeted variants of HBx that are confined to either the nucleus or the cytoplasm due to either the nuclear localization signal (NLS) or the nuclear export signal (NES) tags, respectively. Intriguingly, immunostaining analysis indicated that the subcellular localization of HBx is primarily influenced by its abundance; HBx is confined to the nucleus at low levels but is usually detected in the cytoplasm at high levels. Importantly, HBx, whether re-targeted by either the NLS or NES tag, stimulates viral genome replication to a level comparable to that of the wild-type. Furthermore, similar to the wild-type, the stimulation of viral genome replication by the re-targeted HBx occurred at the transcription level. Thus, we concluded that the stimulation of viral genome replication by HBx is linked to both nuclear and cytoplasmic HBx, although the underlying mechanism of stimulation most likely differs.