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BACKGROUND This study evaluated the effects of the probiotic Bifidobacterium animalis subsp. lactis HN019 (HN019) in the development of experimental periodontitis (EP) in rats submitted to chemoterapy (5-fluorouracil - 5-FU). METHODS Eighty male rats were divided in the following groups: control (C); treated with 5-FU (5FU) (60 mg/kg at day 30 and 40 mg/kg at day 32); treated with PROB (HN019) (daily, for 44 days, starting at day 0); treatment with 5-FU and PROB (5FU-HN019); only EP (EP) (ligature placed on lower first molars at day 30, maintained for 14 days); EP and treatment with 5-FU (EP-5FU); EP and treatment with PROB (EP-HN019); and EP and treatment with 5-FU and PROB (EP-5FU-HN019). Euthanasia occurred at day 44. Morphometric, histomorphometric, microtomographic, immunohistochemical, immunoenzymatic and gene expressions analyzes were performed. The data obtained were statistically analyzed (p<0.05). RESULTS The EP-5FU-HN019 group showed less bone and connective tissue loss when compared to EP-5FU group, while EP-HN019 and EP-5FU-HN019 groups had greater bone volume than EP and EP-5FU groups, respectively (p<0.05). A decrease in immunostaining for TRAP and RANKL, an increase for OPG and lower IL-1 β levels were observed in EP-5FU-HN019 group, when compared to EP-5FU group (p<0.0001). Probiotic therapy led to an increase in the proportions of B. lactis in the feces (p = 0.0018), but not in the biofilm, and reduced the expression of F. nucleatum and P. intermedia in the biofilm (p<0.0001). CONCLUSION B. lactis HN019 reduced the severity of EP in rats submitted to chemotherapy, modulating immunoinflammatory parameters in periodontal tissues and reducing periodontopathogens expression on biofilm in rats submitted to chemotherapy. This article is protected by copyright. All rights reserved.