Bamboo fiber cell walls are crucial mechanical load-bearing units in plants, significantly influencing their physical and mechanical properties. This study explored the multi-layered structure and microfibril orientation of typical moso bamboo fiber with two broad layers. The microfibrils in the primary cell wall exhibited a disordered mesh-like structure with a microfibril angle (MFA) of 90°. The alternating narrow and broad layers in the secondary walls constituted the growth cycle layer together. Within this structure, the microfibrils were oriented in both Z-helices and S-helices. In the narrow layers, the MFA transitioned gradually from outer to inner layers, ranging from approximately 70° to 30°. Conversely, the broad layers had smaller MFA, ranging from about 5° to 20°. Finally, a typical model of multi-layered microfibril orientation of 4-year-old moso bamboo fiber cells was proposed.