Purpose <p>To evaluate the effects of mixed reality (MR)–assisted navigation compared with conventional manual positioning on operative time and tunnel positioning accuracy during anterior cruciate ligament reconstruction (ACLR) using 3D-printed knee models. In particular, this study aimed to determine whether MR navigation can reduce the accuracy gap between senior and junior surgeons.</p> Methods <p>Computed tomography (CT) data from 20 patients with anterior cruciate ligament injury were randomly selected to generate standardized 3D-printed knee models. One senior surgeon and one junior surgeon independently performed femoral and tibial tunnel drilling using either MR-assisted navigation (N) or conventional manual positioning (C). Based on surgeon experience (H/L) and positioning technique (N/C), four groups were established: HN, HC, LN, and LC (n = 5 per group). Operative time was recorded from the initiation of tunnel positioning to completion of tunnel drilling. Postoperative three-dimensional CT scans were used to measure the actual tunnel locations. The absolute percentage deviation between the actual and ideal tunnel coordinates (|Δ%|) was calculated using the Bernard quadrant method for femoral tunnels and the Staubli–Rauschning method for tibial tunnels. Two-way analysis of variance (ANOVA) was used to evaluate the main effects and interaction effects, followed by Fisher’s least significant difference (LSD) post hoc comparisons.</p> Results <p>In the tibial mediolateral (ML) direction, both positioning technique and surgeon experience demonstrated significant main effects, with a significant interaction effect (<i>P</i> &lt; 0.001). MR navigation significantly reduced positioning errors in junior surgeons, bringing their accuracy close to that of senior surgeons. In the tibial anteroposterior (AP) direction, the main effect of navigation was significant (<i>P</i> = 0.009), whereas the interaction effect was not significant. In the femoral D direction, positioning accuracy was primarily influenced by surgeon experience (<i>P</i> = 0.011), whereas only a trend toward improvement was observed in the femoral H direction. Operative time analysis demonstrated a significant interaction effect (<i>P</i> &lt; 0.001): MR navigation significantly prolonged operative time in senior surgeons but did not significantly increase operative time in junior surgeons.</p> Conclusion <p>MR-assisted navigation significantly improves femoral and tibial tunnel positioning accuracy, particularly in the tibial ML direction, and partially reduces the accuracy gap between senior and junior surgeons. Importantly, the improvement in accuracy among junior surgeons was not accompanied by an increase in operative time. These findings suggest that MR navigation may serve as a valuable educational and training tool; however, further clinical studies are required to validate its long-term clinical effectiveness.</p>