SPLUNC1 is a 256 amino acid protein that belongs to the bactericidal permeability-increasing (BPI)-fold containing family A. SPLUNC1 is the most abundant gene in the upper airways and regulates Na+ absorption and has anti-microbial activity (1). The Burkholderia cepacia complex (Bcc) is a group of closely related species known to cause severe infections in people with CF. The group currently comprises of at least 17 genetically distinct species. Species within Bcc differ in virulence, persistence and transmissibility and these bacteria are highly resistant to antibiotics (2). Of the Bcc group, B. cenocepacia and B. multivorans are the most clinically relevant as they are most commonly associated with increased mortality, epidemics and ‘cepacia’ syndrome in CF patients (2, 3, 4).Using a 96 well plate assay we demonstrated antimicrobial and antibiofilm activity of recombinant human SPLUNC1 against Bcc strains grown alone and in combination with polymixin B, an antibiotic used to treat resistant Gram negative infections. The growth of the ET-12 epidemic strain B. cenocepacia J2315 was inhibited by > 30 ug/ml SPLUNC1 (p<0.001). Polymixin B alone did not affect the growth of J2315. However, when SPLUNC1 and varying concentrations of polymixin B were combined the growth of J2315 was significantly inhibited even further compared to treatment with SPLUNC1 alone (p<0.001). This combination also lowered the MIC of SPLUNC1 to 3 ug/ml indicating synergism between the two proteins. This inhibition was also observed with three clinical Bcc isolates, B. cenocepacia AU21968 (p<0.001), B. cepacia AU28001 (p<0.05) and B. multivorans AU27629 (p<0.05). S18, which is an n-terminal SPLUNC1 derived peptide, did not effect the growth of J2315 alone, however, in combination with 10 ug/ml polymixin B, the growth of J2315 was significantly inhibited (p<0.01). Results indicate that S18 was not as effective against J2315 as SPLUNC1. SPLUNC1 also decreased J2315 biofilm formation (p<0.001) where as a SPLUNC1 mutant missing two alpha-helical regions, did not significantly affect biofilm formation. Neither SPLUNC1 nor S18 had any effect on the growth on the Gram positive strain Staphylococcus aureus, indicating that SPLUNC1 is only effect against Gram negative bacteria. We conclude that splunc1 has antimicrobial activity and also had the ability to potentiate the effects of polymixin B. Studies to better understand SPLUNCs mode of action are ongoing. This data expands on the emerging picture of SPLUNC1 as a multifunctional protein and may provide an alternative approach for treating Bcc infections. Funded by NIH HL108927