Golgi α‐mannosidase II is an enzyme that processes the intermediate oligosaccharide Gn1M5Gn2 to Gn1M3Gn2 during biosynthesis of N‐glycans. Previously, we isolated a cDNA encoding a protein homologous to α‐mannosidase II and designated it α‐mannosidase IIx. Here, we show by immunocytochemistry that α‐mannosidase IIx resides in the Golgi in HeLa cells. When coexpressed with α‐mannosidase II, α‐mannosidase IIx colocalizes with α‐mannosidase II in COS cells. A protein A fusion of the catalytic domain of α‐mannosidase IIx hydrolyzes a synthetic substrate, 4‐umbelliferyl‐α‐d‐mannoside, and this activity is inhibited by swainsonine. [3H]glucosamine‐labeled Chinese hamster ovary cells overexpressing α‐mannosidase IIx show a reduction of M6Gn2 and an accumulation of M4Gn2. Structural analysis identified M4Gn2 to be Manα1→6(Manα1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc. The results suggest that α‐mannosidase IIx hydrolyzes two peripheral Manα1→6 and Manα1→3 residues from [(Manα1→6)(Manα1→3)Manα1→6](Manα1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc, during N‐glycan processing.