Abstract This study aimed to investigate whether two acquisition parameters, voxel size and filter thickness, used in a micro-computed tomography (micro-CT) scan, together with the examiner’s experience, influence the outcome of bone repair analysis in an experimental model. Bone defects were created in rat tibiae and scanned using two voxel sizes of 6- or 12-µm and two aluminum filter thickness of 0.5- or 1-mm. Then, bone volume fraction (BV/TV) and trabecular thickness (Tb.Th) were analyzed twice by two groups of operators: experienced and inexperienced examiners. For BV/TV, no significant differences were found between scanning voxel sizes of 6 and 12 µm for the experienced examiners; however, for the inexperienced examiners, the analysis performed using a 12-µm voxel size resulted in higher BV/TV values (32.4 and 32.9) than those acquired using a 6-µm voxel size (25.4 and 24.8) (p < 0.05). For Tb.Th, no significant differences between the analyses performed by experienced and inexperienced groups were observed when using the 6-µm voxel size. However, inexperienced examiners’ analysis revealed higher Tb.Th values when using the 12-µm voxel size compared with 6 µm (0.05 vs. 0.03, p < 0.05). Filter thickness had no influence on the results of any group. In conclusion, voxel size and operator experience affected the measured Tb.Th and BV/TV of a region with new bone formation. Operator experience in micro-CT analysis is more critical for BV/TV than for Tb.Th, whereas voxel size significantly affects Tb.Th evaluation. Operators in the initial phases of research training should be calibrated for bone assessments.