Objective To investigate the application effect of negative pressure lithotomy combined with renal upper calyx puncture in percutaneous nephrolithotomy (PCNL).Methods A total of 86 patients with close calculi load in PCNL in Xianyang First People′s Hospital from September 2019 to August 2021 were selected as the study objects. Patients were divided into observation group and control group by random number table method, with 43 cases in each group. The observation group was treated with negative pressure lithotomy combined with renal upper calyceal puncture during PCNL operation, while the control group was treated with conventional tear sheath puncture through renal middle or lower calyceal puncture. The perioperative related indexes, renal function and oxidative stress related indexes and the occurrence of intraoperative and postoperative complications were compared between the two groups.Results The operative time, intraoperative blood loss, intraoperative stone removal time, intraoperative renal pelvis pressure and postoperative hospital stay were significantly reduced in the observation group compared with the control group, and the stone clearance rate was significantly increased (all P<0.05). The levels of renal function and oxidative stress indexes such as serum creatinine (Scr), urea nitrogen (BUN), cortisol (COR) and norepinephrine (NE) in both groups were significantly increased 7 days after surgery compared with those before surgery (all P<0.05), and the levels of renal function and oxidative stress indexes in the observation group were significantly lower than those in the control group (all P<0.05). There was no significant difference in the incidence of intraoperative and postoperative complications between the observation group and the control group (all P>0.05).Conclusions The application of negative pressure lithotomy combined with renal upper calyceal puncture in PCNL can not only effectively shorten the operation time, reduce intraoperative injury and intrarenal pelvis pressure, improve the lithotomy rate, but also effectively reduce the kidney injury and oxidative stress reaction of patients, and has a tendency to reduce the occurrence of complications, which is more conducive to the postoperative recovery of patients.