The study aimed to investigate the expression level of POU5F1B in non-small cell lung cancer (NSCLC) patients’ serum, tissues, and cell lines and to identify its potential molecular mechanism in NSCLC pathogenesis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was employed to detect POU5F1B expression. The associations between POU5F1B expression and age, gender, body mass index (BMI), smoking, tumor-node-metastasis (TNM) stage, type of tissues, and lymph node metastasis were analyzed. Furthermore, changes in the proliferation and apoptosis of lung cancer cells were observed after down-regulation of POU5F1B. The results demonstrated a significant increase in POU5F1B expression in NSCLC serum and cancer tissues with lymph node metastasis as well as in lung cancer cell lines. POU5F1B expression was found to be related to TNM stage, smoking, and lymph node metastasis in NSCLC patients and was an independent risk factor that affected NSCLC prognosis. Knockdown of POU5F1B reduced lung cancer cell proliferation and increased apoptosis activity. These findings suggest that POU5F1B promotes NSCLC development by promoting cell proliferation and inhibiting apoptosis.