Dilute acid pretreatment (DAP) is an attractive method to overcome the natural recalcitrance of lignocellulosic materials, the enzymatic conversion of which can be improved significantly. Therefore, lignocellulosic biomass can be converted to biofuels with a higher efficiency. However, the mechanism of cell wall deconstruction during DAP on sub-cell level is still unclear. In this study, the topochemical changes of Pinus massonianafiber cell walls after DAP was investigated using confocal Raman microscopy combined with principal component analysis and cluster analysis. The samples were presented with specific distribution in the first and second principal component space, which were with cumulative contribution of 94.61%. The accurate average Raman spectra from different lamellas of cell walls were obtained by cluster analysis. With Raman imaging combined, it was observed that the high-lignified cell corner (CC) was with a high concentration of lignin and the low-lignified secondary wall (SW) was with a high concentration of carbohydrates. The heterogeneity of cell wall deconstruction in Pinus Massoniana during DAP was alsovisualized; the compact structure of raw materials was disrupted. The Raman intensity of typical carbohydrates bans at 2890 cm-1 in the SW decreased 26.9% when compared with that for untreated samples, which indicated thatvast carbohydrates were removed from the SW. A certain amount of carbohydrates was also removed from the compound middle lamella (CML), but slight carbohydrates enrichment was observed in the cell corner. Lignin redistributed during DAP and therefore the Raman intensity of lignin in cell corner was enhanced. The removal of carbohydrates (mainly hemicelluloses) and the lignin redistribution can improve the enzymatic hydrolysis by increasing the cellulose accessibility. The present study not only provides a convenient and efficient method for investigating topochemistry of fiber cell walls, but also brings new insights into studying the high-value conversion of lignocellulosic biomass.