PNGase F works by cleaving between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins and glycopeptides.

It is able to deglycosylate in the absence of denaturants, but needs extensive incubation and larger amounts of the enzyme to cleave native proteins.

It cleaves all asparagine-linked complex, hybrid, or high mannose oligosaccharides unless the core GlcNAc contains an alpha 1,3- fucose.

PNGase F requires a minimum of two GlcNAc oligosaccharide residues attached to the asparagine in order for catalysis to occur.

The crystal structure of PNGase F from flavobacterium miningosepticum, with 1.8 Å resolution, was found to be folded in two domains, each with an eight-stranded antiparallel β barrel, or jelly roll, configuration.