This preclinical study aimed to investigate the role of NF-κB in early and late radiogenic sequelae of urinary bladder dysfunction in mice. Thalidomide was applied either during the early or late response phase to determine potential effects of NF-κB inhibition on functional bladder impairment. After pelvic irradiation on day 0, female C3H/Neu mice were observed over a period of 360 days and radiation response was evaluated for alterations in bladder functionality and NF-κB activation. Functionality was determined in graded dose experiments (14-24 Gy) and assessed by micturition frequency analysis (MFA) and transurethral cystotonometry (TC) to reveal alterations in voiding and volume. The induction of the NF-κB proteins p50 and p65 was evaluated by immunohistochemistry in response to a single dose of 23 Gy (ED90). Thalidomide (100 mg/kg/day) was applied intraperitoneally in three treatment groups: daily from day 1-15, daily from day 16-30 or in 2-day-intervals from day 150-180. Immunohistochemical analysis showed a biphasic activation of p50 and p65 during the early radiation cystitis (ERC) phase (day 1-30). After a transient decrease, p50, but not p65, was reactivated permanently leading to increased levels which suggest an occurrence of chronic inflammation in correlation to functional impairment. Both early thalidomide treatments reduced NF-κB activation and shifted the ED50 value for ERC and late radiation sequelae (LRS) to higher doses. These data clearly demonstrate the involvement of NF-κB signaling in the pathogenesis of radiation-induced urinary bladder dysfunction. Additionally, this study emphasizes that biological targeting of early radiogenic processes has enormous impact on chronic symptoms. The late administration of thalidomide showed no significant effect on functionality.