The increasing demand for sustainable architecture has led to a growing interest in wood structures. Hence, ensuring their structural stability and strength performance is an imperative. This study investigated the nail bearing strength and withdrawal resistance of mixed cross-laminated timber (CLT) using Japanese larch and yellow poplar layers. The mixed CLT was composed of three larch laminas (major) and two yellow poplar laminas (minor). The bearing strength of the mixed CLT decreased as the ratio of the thickness of the minor lamina to nail depth increased. The nail withdrawal resistance differed in the penetration and axial directions of the laminas. In the direction perpendicular to the grain, the withdrawal resistance load of the yellow poplar lamina was measured to be 1.45-times that of the larch lamina. The withdrawal resistance of the mixed CLT with the yellow poplar layer was 17% higher than that with larch. Therefore, the length of the nail used for the mixed CLT should be selected based on the thickness of the minor lamina to achieve efficient bearing and withdrawal resistance of the nail connection.