This study investigated the accuracy of positioning and irradiation targeting for multiple off-isocenter targets in intracranial image-guided radiation therapy (IGRT). A phantom with nine circular targets was created to evaluate both accuracies. First, the central point of the isocenter target was positioned with a combination of an ExacTrac x-ray (ETX) and a 6D couch. The positioning accuracy was determined from the deviations of coordinates of the central point in each target obtained from the kV-cone beam computed tomography (kV-CBCT) for IGRT and the planning CT. Similarly, the irradiation targeting accuracy was evaluated from the deviations of the coordinates between the central point of each target and the central point of each multi-leaf collimator (MLC) field for multiple targets. Secondly, the 6D couch was intentionally rotated together with both roll and pitch angles of 0.5° and 1° at the isocenter and similarly the deviations were evaluated. The positioning accuracy for all targets was less than 1 mm after 6D positioning corrections. The irradiation targeting accuracy was up to 1.3 mm in the anteroposterior (AP) direction for a target 87 mm away from isocenter. For the 6D couch rotations with both roll and pitch angles of 0.5° and 1°, the positioning accuracy was up to 1.0 mm and 2.3 mm in the AP direction for the target 87 mm away from the isocenter, respectively. The irradiation targeting accuracy was up to 2.1 mm and 2.6 mm in the AP direction for the target 87 mm away from the isocenter, respectively. The off-isocenter irradiation targeting accuracy became worse than the positioning accuracy. Both off-isocenter accuracies worsened in proportion to rotation angles and the distance from the isocenter to the targets. It is necessary to examine the set-up margin for off-isocenter multiple targets at each institution because irradiation targeting accuracy is peculiar to the linac machine.