Phytases are valuable industrial enzymes widely used in animal feed production, and when expressed in yeast, they undergo glycosylation. Herein, heterologous expression in Pichia pastoris and biochemical characterisation of glycosylated and deglycosylated forms of a novel phytase from Cronobacter turicensis belonging to the histidine acid phosphatase family were successfully carried out. Mutants with deleted N-glycosylation sites (N136, N171, and N202) were constructed by site-directed mutagenesis and characterised. Deglycosylation greatly changed the resistance of phytase to proteolysis, acidic pH, and temperature, but did not significantly affect other biochemical properties. High specific activity (1705 U/mg), Km (173 μM), and Vmax (2778 μmol min−1 mg−1), as well as the optimum temperature (50 °C) and pH (4.5) of the enzyme did not depend on the degree of glycosylation. The degree of change in resistance of phytase depended on the position of the N-glycosylation site on the protein globule. The removal of glycan at the N202 site located in the highly flexible region of the protein had the greatest effect on the enzyme characteristics and led to a decrease in resistance of phytase to pepsin and trypsin by 73% and 87%, respectively. Conversely, the glycosylated enzyme exhibited strong resistance to pepsin, trypsin, and acidic pH, and increased thermostability. It has been shown that N-glycosylation occurring in P. pastoris served as a powerful tool for improving the biochemical properties of recombinant phytase and made it promising for use as an animal feed additive.