ABSTRACT Penetrated mortise-tenon joints (PMJs) are typical wood-to-wood connections commonly used in traditional Chinese timber structures. They play a crucial role in the structural behavior of timber constructions. This study derived a method of theoretical estimation for the rotational behavior of PMJs, which is unique and more comprehensive in that it takes both movement of the rotation center and bending deformation of the tenon into consideration. In addition, it experimentally and numerically validated the theoretical model, and quantitatively analyzed the position changes of the rotation center and the effect of the bending deformation. On this base, simplified calculation formulas are proposed for the prediction of equivalent elastic stiffness and peak moment of PMJs, and then applied for the calculation of the lateral stiffness of timber frames. As a result, the predicted lateral stiffness shows good agreement with test results, demonstrating the validity of the estimation method derived and its applicability to the structural analysis. The results of this study show that the rotation center of the tenon is not stationary, and its moving range in the horizontal direction is much larger than that in the vertical direction. Another pattern found is that the bending deformation of the smaller tenon counteracts more than 20% of the compressive deformation caused by the rigid-body motion, and therefore must be considered when analyzing the rotational behavior of PMJs.