Glycans (i.e. oligosaccharide chains attached to cellular proteins and lipids) are crucial for nearly all aspects of life, including the development of multicellular organisms. They come in multiple forms, and much of this diversity between molecules, cells, and tissues is generated by Golgi-resident glycosidases and glycosyltransferases. However, their exact mode of functioning in glycan processing is currently unclear. Here we investigate the supramolecular organization of the N-glycosylation pathway in live cells by utilizing the bimolecular fluorescence complementation approach. We show that all four N-glycosylation enzymes tested (β-1,2-N-acetylglucosaminyltransferase I, β-1,2-N-acetylglucosaminyltransferase II, 1,4-galactosyltransferase I, and α-2,6-sialyltransferase I) form Golgi-localized homodimers. Intriguingly, the same enzymes also formed two distinct and functionally relevant heterodimers between the medial Golgi enzymes β-1,2-N-acetylglucosaminyltransferase I and β-1,2-N-acetylglucosaminyltransferase II and the trans-Golgi enzymes 1,4-galactosyltransferase I and α-2,6-sialyltransferase I. Given their strict Golgi localization and sequential order of function, the two heterodimeric complexes are probably responsible for the processing and maturation of N-glycans in live cells.