Researchers have put much attention on the utilization of biomaterial resources for sustainable development. The change of fossil-based economy to bio-economy urges to consider using bio-based materials. To replace fossil-based products, bioconversion of biomaterial to develop value-added products, i.e., biochemical, bioethanol, is very important. Wood is the major source of biomaterial on the earth. Size-reduction of wood is the prerequisite to use it in the downstream process. The quality of the wood powder is dependent on the milling technology. The change of micro-and ultrastructural during milling affects the conversion pathway. The opening pore and cellulose exposure are beneficial to enzymatic hydrolysis leading to more glucose production. In this study, green milling technology (multi-blade shaft mill (MBSM)) was used to produce powder from the wet and dry pine (Pinus sylvestris L.) logs. The cell wall fracture mechanism, cell lumen, pore in the cell wall, and middle lamella of the produced powders will be conducted. The extractive location and parenchyma condition of them will also be investigated. These will provide a better understanding of the structural changes of the produced powders. For the comparison, the powders produced from the hammer mill using a single setting will be studied. This study will help consider the proper utilization of MBSM powder in biorefinery.