The assessment of tumor response to neoadjuvant chemotherapy (NACT) in locally advanced gastric cancer (LAGC) remain challenging. We aimed to explore the potential role of peri-NACT change of the largest lymph node (LN) and primary tumor (P-T) in the prediction of tumor response and patient overall survival (OS) in LAGC. A cohort of LAGC patients who underwent NACT followed by radical surgery from a prospective clinical trial were retrospectively analyzed. The percentage change of the largest LN and P-T from initial to post-NACT Computed Tomography (CT) were measured. Tumor response was defined by the change of LN (LN-response) and P-T (P-T-response), respectively. A multivariate Cox model was constructed to examine if P-T- and LN-determined response had significant predictive ability for OS when adjusting with other possible prognostic factors. Of the 41 patients, 28 (68.3%) was defined as LN-responders to NACT, and 17 (41.5%) patients was defined as P-T-responders. When the cohort was stratified by LN response standard, LN-responders showed a significant longer median OS than LN-nonresponders (p = 0.031, 20.6 vs 16.6 months). When stratified by primary tumor response, no significant difference in OS was observed between P-T-responders and P-T-nonresponders (p = 0.377, 18.5 vs 19.0 months). In the multivariate analysis, number of positive LN (p = 0.004, hazard ratio [HR] = 1.284), recurrence (p = 0.024, HR =3556), LN shrinkage (p = 0.022, HR = 0.930) and LN-response (p = 0.033, HR = 0.008) were observed with independent association with OS. Peri-NACT change of the largest LN could reflect tumor response to NACT, and LN-defined response was useful in predicting the long-term prognosis (OS) of LAGC patients who underwent NACT followed by radical surgery.