Background: The significance of nano-carbon for lymph node staging in radical gastrectomy for gastric cancer (GC) has been confirmed, but studies on its application for GC patients treated with neoadjuvant chemotherapy (NCT) are rare. The purpose of this study was to explore the clinical value of using carbon nanoparticles suspension injections (CNS) to improve the accuracy of lymph node staging (N staging) of NCT for advanced GC. Methods: 160 advanced GC patients receiving preoperative NCT were enrolled, according to the random number generated by computer, the enrolled patients were randomly divided into two groups: experimental group (n=80) and control group (n=80). The experimental group received endoscopic injection of CNS within 24 hours prior to NCT, while the control group received this within 24 hours post NCT and before D2 radical resection. SOX [oxaliplatin: 130 mg/(body surface area, BSA): m(2), first day + S-1: (BSA: <1.25 m(2), 40 mg each time; >= 1.25 to <1.5 m(2), 50 mg each time; >= 1.5 m(2), 60 mg each time), 2 times a day, for 2 weeks] was chosen as the NCT regimen, repeat every 3 weeks, 4 cycles were performed preoperative. Surgery was performed 3 weeks after the end of the 4 cycles of chemotherapy. The staining rate, metastasis rate, metastasis rate of stained lymph nodes, postoperative complication rate, and N staging of the two groups were analyzed and compared. Results: A total of 3,197 lymph nodes were harvested in the experimental group, including 384 metastatic lymph nodes, 1,424 stained lymph nodes, and 210 metastatic stained lymph nodes. The total number of lymph nodes harvested in the control group was 2,565, including 244 metastatic lymph nodes, 796 stained lymph nodes, and 94 metastatic stained lymph nodes. Compared with the control group, a higher rate of stained lymph nodes, a higher total number of lymph nodes, and an increased number of metastatic lymph nodes were detected in the experimental group. Conclusions: The application of CNS before NCT in patients with advanced GC can minimize lymph node staging bias after NCT and improve its accuracy.