The human UBE3A gene encodes three isoforms of Ubiquitin E3 ligase A, which differ in their N-terminal sequence, abundance, and localization. The most abundant isoforms are the short human UBE3A isoform 1 (hUBE3A-Iso1) (80%) and the long hUBE3A-Iso3 (17%), which are highly conserved across mammals. In mouse and human neurons, UBE3A levels are most prominent in the nucleus, which is largely attributed to the abundant short hUBE3A-Iso1 which is enriched in the nucleus. In mice, the cytosolic UBE3A staining is predominantly the result of cytosolically enriched long m(ouse)UBE3A-Iso2. Despite the high sequence conservation between the long mUBE3A-Iso2 and its human ortholog hUBE3A-Iso3, we previously reported nuclear enrichment for hUBE3A-Iso3. This finding was not only surprising, but also seemed at odds with the substantial cytosolic UBE3A staining that is observed in human neurons. Here, we revisited the localization of hUBE3A-Iso3, as well as an Angelman syndrome-associated variant in the N-terminal sequence of hUBE3A-Iso3. We now conclude that hUBE3A-Iso3 isoform and its mouse ortholog mUBE3A-Iso2 show in fact a very similar mostly cytosolic localization. Our previous, erroneous, conclusions are explained by the finding that N-terminal amino acid substitutions affect the localization of these two orthologs in different ways. Specifically, amino acid substitutions at the p.Met21 position of hUBE3A-Iso3 result in a partial shift of the hUBE3A-Iso3 isoform from the cytosol to the nucleus.