The influence of calcination temperature on ZrO2 and its catalytic activity in glucose conversion was studied in this research. It shows that different structure of ZrO2 can be obtained by tuning calcination temperature, which results in the various surface catalytic properties. Quantitative evaluation of acidity by NH3-TPD and solid-state NMR spectroscopy shows that ZrO2 calcined at 300 degrees C, which is in amorphous state and has a higher BET surface area, possesses more Bronsted and Lewis acid sites than ZrO2 samples calcined at other temperatures. Amorphous ZrO2 shows a better catalytic performance in glucose conversion, nearly 100% glucose conversion with an HMF selectivity of about 40%. Increasing calcination temperature leads to a result of sintering, crystallizing, and pore collapsing of ZrO2. There is a distinct decrease in Bronsted acid sites, along with a decrease of the total number of acid sites in ZrO2 as calcination temperature increases. At the same time, a new type of Lewis acid appears at a downfield shift, resulting in different reaction rates.