Nitration of acetyl guaiacol, one typical aromatic nitration, is highly exothermic and extremely fast. Better control and high efficiency can be achieved in the microreactor due to its enhanced mixing and heat transfer rates. In this study, nitration of acetyl guaiacol was carried out in a microreactor using nitric acid-acetic acid as nitrating agent. The nitration kinetics was first investigated, and a kinetic model was established and revealed good prediction of experimental results at higher temperatures. Effects of molar ratio of nitric acid-acetyl guaiacol, residence time, temperature, and nitric acid concentration on the reaction were studied in detail. Under optimized condition, 90.7% yield of desired product, 5-nitroguaiacol, was achieved with 40% of nitric acid concentration, nitric acid-acetyl guaiacol molar ratio of 2.6, reaction temperature of 120 °C, and residence time of 2 min. Compared to traditional batch reactor, microreactor showed the advantages of higher yield and selectivity, much shorter reaction time, and less use of nitric acid.