The impact of desilication on the performance of a series of alkali-treated NaY zeolites impregnated with 5 wt.% of magnesium for glucose isomerisation into fructose has been studied. Desilication at different NaOH concentrations increases the mesoporous volume and external surface area, without compromising microporosity and crystallinity. The observed reduction of the microporous volume due to magnesium impregnation was found to decrease for the alkali-treated zeolites. Higher density and strength of basic sites and stronger magnesium-support interaction were also achieved with the treatment. These improved properties resulted in a significant increase of both glucose conversion and fructose yield on the magnesium-doped desilicated zeolites. Glucose conversion continuously increases with desilication (28–51%), whereas fructose yield passes through a maximum (35%) at low desilication levels. Among the prepared desilicated samples, low-severity alkali-treated zeolites also show lower deactivation in consecutive reaction runs, as well as superior regeneration behaviour. Thus, hierarchical NaY zeolites impregnated with magnesium could be favourably used for glucose isomerisation into fructose if suitable alkaline treatment conditions are selected, with low-severity treated NaY zeolites being the best choice. Higher fructose productivities were achieved for the low-severity desilicated zeolites than for higher magnesium content NaY zeolites reported previously, leading to a lower Mg requirement.