Ethylene glycol (EG)-intercalated layered double hydroxides (LDHs) with various Ni/Al molar ratios were prepared by a microwave-assisted ion exchange method. In comparison with the conventional ion exchange method, this method can achieve a more ordered layered structure and reduce the ion exchange time from 12 h to 30 min. The as-prepared EG-intercalated LDHs were used as esterification catalysts to remove naphthenic acids in crude oil. It is found that EG-intercalated LDHs may act as nanosized intracrystalline catalytic microreactors, attributed to the preintercalated EG which reduces the diffusion resistance, the enlarged interlayer space which facilitates the access of naphthenic acids, and metal catalytic sites in the layer. The confined interlayer space enhances the collision between reactants and accelerates the reaction. Therefore, EG-intercalated LDHs exhibit several times higher acid removal ratio and EG conversion than nitrate LDHs. The increasing Ni/Al ratio increases the amount of catalytic sites but decreases the intercalated EG content, which imposes opposite effects on the acid removal ratio. The catalysts and crude oil after reaction were characterized, and the possible process of the intracrystalline catalytic esterification was proposed.