Abstract Tobacco leaf consists of numerous cells with irregular size and wavy shapes, which determine the leaf’s nonlinear macro behavior. In this paper, the Scanning Electron Microscope (SEM) has been utilized to characterize the cellular microstructures of tobacco leaf tissue, and uniaxial tensile tests have been performed to obtain the mechanical behaviors, characterized by two linear responses separated by a nonlinear transitional phase. Following the SEM characterization of cellular microstructure, a finite element based Representative Volume Element (RVE) was proposed for the first time, and the Young’s moduli of cellular wall and protoplast have been identified in terms of the averaged test data and the RVE model. Further, in terms of the verified RVE model, the connection between the unique hardening response at macro level and the cellular-level deformations has been explored in detail. This study also illustrates the significance of loading direction effects due to the variation of the loading path formulation, which is determined by the intrinsic cellular arrangement.