The objective of this study was to analyze the stiffness variation of maize fiber bundles. Tensile modulus of fiber bundle was calculated by using a multiscale simulation. Fiber bundle cross section was reconstructed by the skeletonization method. The influences of the cell wall thickness, the microfibril angle (MFA) in S2 layer and the fiber diameter on the tensile modulus of retted maize bundles were determined. Numerical results showed that the MFA in S2 layer and the cell wall thickness in vascular bundle sheath (VBS) are the main factors affecting the tensile stiffness of maize bundle. When fiber diameter is fixed, the large variation of tensile modulus is attributed to the small MFA in S2 layer and the big difference in cell wall thickness of VBS. For fiber bundles in the skin group, the decreased tendency of tensile modulus is attributed to the decreased density of VBS when fiber diameter increases. However, for fiber bundles in the core group, the combined effects of decreased density of VBS and increased area ratio of Sv (transverse sectional area occupied by vessels and phloem tissue in fiber bundle) contribute more than 60% to the decreased tensile modulus. Compared to the increased area ratio of Sv, the decreased density of VBS exerts a greater influence on the decrease in stiffness. These findings can enhance our understanding of the large variation of natural fiber stiffness.