Objective: The purpose of this study was to develop a phantom-based experimental calibration method to minimize the reconstruction artifacts for the geometric misalignments of the digital tomosynthesis prototype. Methods: A calibration phantom with ten fiducial markers was designed. Using this calibration phantom, the projection matrices of an experimental digital tomosynthesis prototype were acquired from each projection view under a series of misalignment conditions. The American College of Radiology mammography phantom was imaged and reconstructed with and without using the correction of the corresponding calibration projection matrices. The effectiveness of the calibration technique was then quantitatively analyzed through comparison of the calibrated and uncalibrated images. Results: As the isocenter horizontal-shift increases, the reconstruction artifacts become clearly distinguishable. Using the calibration technique, the reconstruction artifacts resulting from the isocenter horizontal-shift were effectively minimized for the prototype. Conclusions: For the specific experimental conditions utilized in this study, the phantom-based calibration method effectively reduced reconstruction artifacts for the prototype investigated in this study. The calibration method holds potential to benefit other tomosynthesis applications. © 2012-IOS Press and the authors. All rights reserved.