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Burkholderia cenocepacia is an opportunistic pathogen prevalent in cystic fibrosis patients, which is particularly difficult to treat, causing chronic and eventually fatal infections. The lack of effective treatment options makes evident the need to develop alternative therapeutic or prophylactic approaches. Vaccines, and live attenuated vaccines, are an unexplored avenue to treat B. cenocepacia infections. Here we constructed and characterized a B. cenocepacia tonB mutant strain, which was unable to actively transport iron, to test whether this single gene deletion mutant (strain renamed GAP001) protected against an acute respiratory B. cenocepacia lethal infection. Here we show that the mutant strain GAP001 is attenuated, and effective at protecting against B. cenocepacia challenge. Intranasal administration of GAP001 to BALB/c mice resulted in almost complete survival with high degree of bacterial clearance.