UDP‐N‐acetylglucosamine:α(1,6)‐D‐mannoside β(1,6)‐N‐acetylglucosaminyltransferase (GnT‐V, Mgat5) functions in the biosynthesis of N‐linked glycans and is transcriptionally upregulated by oncogene signaling. We report here the cloning and characterization of a human cDNA encoding a distinct enzyme with related substrate specificity, termed GnT‐VB, which is predicted to have 53% similarity to the original amino acid sequence of GnT‐V(A). Transient expression of GnT‐VB cDNA in COS7 cells yielded significant increases of activity toward GnT‐VA acceptors, including synthetic saccharides and N‐linked glycopeptides, with some differences in specificity. Unlike GnT‐VA, GnT‐VB required divalent cation for full activity. EST databases showed expression of a 6 bp (+) splice isoform of GnT‐VB; when expressed, this enzyme showed significantly reduced activity. CHO Lec4 cells, which do not express GnT‐VA or B activity, lack synthesis of the N‐linked β(1,6) branch, and do not bind L‐phytohemagglutinin (L‐PHA), were transfected with GnT‐VB or GnT‐VA; both then bound significant amounts of L‐PHA, demonstrating that both enzymes synthesized N‐linked β(1,6) branched glycans in vivo. Real‐time polymerase chain reaction results showed that GnT‐VB mRNA was highly expressed in brain and testis, with lesser levels in other tissues, while human GnT‐VA showed a more general expression, but with low levels in brain and no expression in skeletal muscle.