Background: To evaluate the safety and efficacy of intrawound vancomycin powder in reducing surgical site infections (SSIs) after spine surgery. Design: A prospective, double-blind, randomized controlled study. Participants: Patients who underwent posterior lumbar interbody fusion (PLIF) surgery from May 2021 to September 2022. Methods: Patients who underwent PLIF surgery between May 2021 and September 2022 were included. Participants were randomized to the vancomycin treatment or control groups using block randomization (block size 4). Except for baseline and surgical data, the plasma levels of white blood cells, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), aspartate aminotransferase, alanine aminotransferase, and serum vancomycin concentration in the groups were analyzed on postoperative days (PODs) 1, 3, and 5. Vancomycin concentration was measured daily until the drainage tubes were removed. The primary outcomes were the 90-day vancomycin-related adverse reactions and SSI rates. Secondary outcomes were perioperative hematological parameters and vancomycin serum (drain) concentrations. Results: A total of 156 participants (78 each in each group) were analyzed by an independent researcher. The follow-up rate was 91%. All participants were followed up for at least 90 days. The 90-day SSI rate in the vancomycin group was 1.3% (1/78), comprising one case of superficial infection. The SSI rate in the control group was 10.3% (8/78), comprising seven cases of superficial infection and one case of deep infection. Compared with that in the control group, the SSI rate in the vancomycin group was decreased by 87.5%, with a statistically significant difference (RR=0.125, 95% CI=0.016–0.976). Additionally, the vancomycin group demonstrated a statistically significant decrease in serum ESR on POD 3 ( P =0.039) and CRP on POD 5 ( P =0.024) compared to the control group. The local plasma concentration of vancomycin remained elevated for at least 4 days postoperatively, while the serum concentration of vancomycin remained low. Vancomycin-associated adverse reactions were not observed. Conclusion: Intrawound application of vancomycin powder is a safe and effective procedure for reducing the risk of SSI during PLIF surgery.