Lignocellulosic biomass offers advantages over corn starch for the production of biofuels, from both economic and environmental standpoints. However, due to the chemical resistance of lignin and complex structure of the plant cell wall, energy-intensive and polluting pretreatments of the biomass are necessary for its fractionation into liquid fuels, materials, and chemicals. Recently, ionic liquids have attracted considerable interest for their ability to dissolve biomass at high temperatures. In this study, the swelling of poplar wood cells during ionic liquid (1-ethyl-3-methylimidazolium acetate) pretreatment was monitored with time-lapse series of autofluorescence images. After a 3 h ionic liquid pretreatment at room temperature, the secondary cell walls were noticeably swollen, indicated by an increase of the cell wall areas ranging from 60% to 100%, depending on the initial cell area. After the ionic liquid pretreatment, the wood sample was rinsed with de-ionized water. The rinsing resulted in an immediate reduction of the secondary cell wall swelling and also an increase of the cell total area (cell wall and lumen). These results contrast with cell area measurements on poplar wood cells exposed only to de-ionized water, that show an initial increase in cell wall area and a complete recovery 3 h after the exposure to de-ionized water.