BACKGROUND POU5F1B, serving as a carcinogen, participates in radiosensitivity of several tumors. However, in esophageal cancer, its potential mechanism and function in regulating radiosensitivity remain unclear. MATERIAL AND METHODS The expression level of POU5F1B was detected in plasma of esophageal tumor patients and cancer cell lines. The effect of POU5F1B knockdown on cell proliferation and colony formation was determined using CCK-8 assay and colony formation assay. Cell apoptosis rate was detected by flow cytometry. RESULTS POU5F1B expression level declined after radiotherapy in the plasma of esophageal cancer patients (p=0.025). Compared with HEEPIC, the level of POU5F1B was upregulated in ECA109 (p<0.01), ECA9706 (p<0.01), KYSE410 (p<0.01), and KYSE510 (p=0.036). The silencing of POU5F1B played a role in inhibiting colony formation. After radiotherapy, the apoptosis rates in the ECA109 with 4Gy si-POU5F1B group and 4Gy si-NC group were 39.1±0.1% and 35.3±0.1%, respectively (p=0.0193). The rate was 21.00±0.1 and 29.1±0.1% (p<0.0072) in the si-NC group and si-POU5F1B group, respectively. For proliferation rate, 4Gy si-POU5F1B ECA109 performed better than 4Gy si-NC. CONCLUSIONS Radiotherapy contributed to the decline in the expression level of POU5F1B in plasma, which was upregulated in ECA109, ECA9706, KYSE410, and KYSE510, but not in HEEPIC. The knockdown of POU5F1B increased the radiosensitivity of esophageal cancer cell lines.