AIM: To investigate the implication of respiratory microbiology on the efficacy of PD-1 inhibitors monotherapy for patients with NSCLC.  METHODS: This study was designed as a retrospective study, fifty-eight patients with previously-treated advanced NSCLC who were received PD-1 monotherapy from October 2018 to October 2021 were included. The PD-1 inhibitors were consisted of camrelizumab, sintilimab and pembrolizumab. Additionally, the basic demographic data, therapeutic efficacy data, survival prognosis and adverse reactions during the PD-1 inhibitors treatment were collected and analyzed through the patients' medical records of the department and the electronic medical record system of the hospital. Furthermore, deep induced sputum specimens of the patients before treatment with PD-1 inhibitor were collected. And the respiratory microbiology of 58 samples were detected using 16S rRNA gene sequencing method. The index of respiratory microbiology α diversity was analyzed, and the correlation analysis was performed with the efficacy and prognosis of patients.RESULTS: A total of 58 patients with advanced NSCLC met the study's screening criteria and were evaluable for efficacy and safety profile. Efficacy data suggested that the objective response rate (ORR) and disease control rate (DCR) of the patients who received PD-1 inhibitors was 19.0%(95%CI: 9.9%-31.4%) and 55.2%(95%CI: 41.5%-68.3%). Furthermore, prognostic data obtained from follow-up indicated that the median PFS of the 58 patients with advanced NSCLC was 3.2 months (95%CI: 2.29-4.11) and the median OS was 10.5 months (95%CI: 5.58-15.43). Regarding the exploratory analysis between efficacy and respiratory microbiology, the 58 patients with NSCLC were divided into high α diversity group (group H) and low α diversity group (group L) according to Shannon diversity index of respiratory microecology detection. And the association analysis suggested that the ORR of patients with group H and group L was 23.3%and 17.9%(P=0.380), respectively. Furthermore, prognostic analysis indicated that the median PFS of patients with group H and group L was 3.8 and 2.8 months, respectively, which was statistically significant (P=0.034). CONCLUSION: PD-1 inhibitors monotherapy demonstrated preliminary efficacy and prognosis as subsequent line treatment for patients with advanced NSCLC. Patients with higher α -diversity of respiratory microbiology might confer a superior prognosis. And the conclusion should be validated in large sample prospective clinical trials subsequently.