In this paper, a novel positioner fixture with a high repeated positioning accuracy and a high stiffness is proposed and investigated. A high-precision end-toothed disc is used to achieve the high repeated positioning accuracy of the designed positioner fixture. The mathematical models of the cumulative error of the tooth pitch, the tooth alignment error and the error of the tooth profile half-angle of the end-toothed disc are analyzed. The allowable tolerance values of the cumulative error of the tooth pitch, the tooth alignment error and the error of the tooth profile half-angle of the end-toothed disc are given. According to the theoretical calculation results, a prototype positioner fixture is fabricated and its repeated positioning accuracy and stiffness are tested. The test results indicate that the stiffness of the proposed positioner fixture is 1050.5 N/μm, which is larger than the previous positioner fixtures of the same type. The repeated positioning accuracy of the proposed positioner fixture in the x, y and z directions are ± 0.48 μm, ± 0.45 μm and ± 0.49 μm, respectively, which is significantly higher than the previous positioner fixtures.