Deconstruction of lignocellulosic plant cell walls to fermentable sugars by biochemical means is impeded by several poorly understood ultrastructural and chemical barriers. Pretreatment is an essential step by altering the morphological and compositional characteristics of biomass to enhance the sugar release during enzymatic hydrolysis. Therefore, getting insight into this field is necessary to improve the conversion of biomass into biofuels. In this review, we highlight our recent understanding on the impact of various promising pretreatments on biomass, with emphasis on the topochemical and ultrastructural changes of plant cell walls that are related to the reduction of recalcitrance and the consequence of saccharification. It will lend support to the scientific research and development with respect to biomass conversion.