During pyrolysis of holocellulose, secondary reactions of the primary pyrolytic products inevitably occur, affecting the final pyrolytic product distribution. Carboxylic acids from primary pyrolysis process have significant interaction effects on both holocellulose and its pyrolytic products, whereas, the interaction mechanisms are still unclear. In the present study, acetic acid is selected as the typical carboxylic acid product, and glycerol is selected to represent the various hydroxyl-based compounds (both holocellulose and pyrolytic products such as anhydrosugars, etc.). The density functional theory (DFT) method is adopted to investigate the interaction mechanisms between them. Calculation results indicate that acetic acid and glycerol have strong interactions, with acetic acid acting as a catalyst for these interactions in two patterns. (I) Acetic acid enhances the dehydration reactions of glycerol with low energy barriers. (II) Acetic acid and glycerol undergo esterification to form an ester intermediate which then decomposes via various reactions. In addition, the decomposition of acetic acid can also be promoted by the catalysis of glycerol in a certain degree. This study reveals the basic interaction mechanisms between carboxylic acids and hydroxyl-based compounds, providing fundamental information to understand the secondary reactions during pyrolysis of holocellulose.