Correlating the structure with nanomechanical property of semicrystalline conjugated-polymer crystal is of essential importance for the performance improvement and design of flexible electronic devices. Although it is well-known that the semicrystalline conjugated-polymer crystal exhibits anisotropic structure owing to the π-π and layer stacking of highly coplanar conjugated backbones, the structure-nanomechanical property relationship is missing. Here, we investigated the axial mechanical anisotropy of the P3HT nanofiber by using thermal shape-fluctuation analysis and a three-point bending test based on atomic force microscopy. Our results show that Young's modulus in the layer-stacking direction (EL) is 1-2 orders of magnitude greater than that in the π-conjugated backbone direction (EB). We attribute this mechanical anisotropy to the π-stacking of the P3HT backbone, but the layer stacking will decrease EL, which weakens the mechanical anisotropy. Moreover, we demonstrated that the P3HT nanofiber shows a loading-rate-independent Young's modulus and deformation-dependent resilience in the layer-stacking direction.