Unlike 5-hydroxytryptamine (5-HT, serotonin) 1 and 5-HT2, the effect of 5-HT3 receptors on tumor cells is poorly understood. We conducted this study to determine whether the perioperative use of 5-HT3 receptor antagonists, which are widely used antiemetics, impacts the recurrence and mortality after lung cancer surgery and related anti-tumor mechanisms. From data on 411 patients, propensity score matching was used to produce 60 1:2 matched pairs of patients, and variables associated with the prognosis after open lung cancer surgery were analyzed. Additionally, the effects of 5-HT3 receptor antagonists were confirmed in vitro on A549 human lung adenocarcinoma cells. Cancer recurrence occurred in 10 (8.2%) and 14 (22.95%) patients (p = 0.005), treated or untreated, with palonosetron or ramosetron. Perioperative usage of palonosetron or ramosetron was also associated with lower recurrence rate after lung cancer surgery (hazard ratio (HR), 0.293; 95% confidence interval (CI) 0.110–0.780, p = 0.0141). Our in vitro experiments also showed that palonosetron and ramosetron inhibited cell proliferation and colony formation and reduced migration, which was associated with autophagic cell death via the extracellular signal-regulated kinase (ERK) pathway. Palonosetron and ramosetron may have anti-tumor potential against lung cancer cells, suggesting the need to consider these drugs as first-choice antiemetics in patients undergoing lung cancer surgery.