Purpose <p>The objective of this study was to explore the feasibility of using the TianJi Robot system for navigated needle positioning in the PCNL procedure in vitro.</p> Methods <p>A pig kidney with a segment of ureter was selected as the in vitro organ model. Iodine contrast agent was infused into the renal pelvis to dilate the renal pelvis and calyx to establish the in vitro hydronephrosis model. Fluoroscopic images were obtained using a 3D C-arm and transferred to the robotic workstation. Three-dimensional reconstructed images were generated and displayed on the monitor screen. The surgeon planned the optimal puncture trajectory on the workstation. After that, the robotic arm was instructed to move to the surgical field. The guiding cannula was placed onto the robotic arm and brought close to the skin. The needle was placed along the guiding cannula. An 18G needle was inserted manually. Finally, a second CBCT scan was performed to validate the needle’s location.</p> Results <p>Three hydronephrosis models were used in the study, and a total of seven target calyces were planned. six target calyces were successfully punctured in one attempt, with a single-puncture success rate of 87.5%.</p> Conclusions <p>A high single-puncture success rate is achieved using the TianJi Robot system for navigated needle positioning in the PCNL procedure in vitro, marking an innovative step forward in urological interventions.</p>