The aim of this study was to evaluate the effect ofLactobacillus delbrueckiisubsp.lactis (L.del)on vaginal microbiota (VM) dysbiosis and vaginal radiation injury in gynecologic cancer patients. The inhibitory effects ofL.delon cervical cancer cells were also studied in vitro. Gynecologic cancer patients receiving radiotherapy were randomized into control andL.delintervention groups. The control group received radiotherapy, while the intervention group received radiotherapy andL.delintervention (1 capsule/day placed into the deep vagina from the first day of radiotherapy until the end of treatment). Vaginal swab samples were collected on the first day pre-treatment and the last day post-treatment. DNA from 54 patients was extracted and assessed by the 16S rRNA sequencing method. Radiotherapy resulted in vaginal microbiome dysbiosis characterized by increased phylogenetic diversity and increased abundance ofBrevundimonas,StreptococcusandPrevotella, but a decreased abundance ofLactobacillus. Level 2 vaginal radiation injury was positively associated with the abundance ofBrevundimonasand gram-negative non-fermenting bacteria. Administration ofL.delattenuated the reduction ofLactobacilluswhile also inhibiting the abundance ofStreptococcusandPrevotella, thereby ameliorating radiotherapy-related vaginal microbiota dysbiosis. CLD inhibited the in vitro proliferation of SiHa cells by altering the expression of BCL2, HPV16-E6, HPV16-E7, IL6, MAP7, BAX, Caspase-3, Caspase-9 and LTF. In conclusion,L. delapplication can alleviate radiation-induced vaginal dysbiosis and restoreLactobacillusdominance of the vaginal microbiome. Moreover, CLD was found to inhibit cell growth and promote the apoptosis of SiHa cells in vitro. The registration number for this clinical trial is ChiCTR1900021784.