ObjectivesRadiation injury is common after radiotherapy for head and neck cancer. Radiotherapy can reshape the immune microenvironment and cause immunosuppression, including dysregulation of immune checkpoints (ICs). However, the relationship between oral ICs expression after radiation and the development of second primary tumors is unclear.MethodsClinical specimens of second primary oral squamous cell carcinoma after radiotherapy (s‐OSCC) and primary OSCC (p‐OSCC) were collected. The expression and prognostic value of PD‐1, VISTA, and TIM‐3 were analyzed using immunohistochemistry. To further clarify the relationship between radiation and ICs alteration, a rat model was constructed to explore the spatiotemporal changes of ICs in the oral mucosa after radiation.ResultsIn carcinoma tissue, the expression of TIM‐3 was higher in s‐OSCC than in p‐OSCC, while the expression of PD‐1 and VISTA was similar between the groups. In para‐carcinoma tissue, the expression of PD‐1, VISTA, and TIM‐3 was higher in s‐OSCC. High ICs expression was associated with poor survival. In the rat model, ICs were locally upregulated in the irradiated tongue. Moreover, there was a bystander effect, in which the ICs were also upregulated in the unirradiated site.ConclusionRadiation may upregulate ICs expression in oral mucosa and contribute to the development of s‐OSCC.