Burkholderia pseudomallei (Bps) is a Gram-negative bacterium that causes melioidosis, an infectious disease of animals and humans. This disease has high incidence in northern and north-eastern parts of Thailand. Successful treatment of melioidosis is difficult due to high intrinsic resistance of Bps to most antibacterial agents. It has been studied that the antimicrobial resistance of this organism may result from poor permeability of the active compounds through porin channels located in the outer membrane (OM) of the bacterium. In previous work, a 38- kDa protein, namely “BpsOmp38”, was isolated from the OM of Bps. A topology prediction and liposome-swelling assay suggested that BpsOmp38 comprises a β-barrel structure and acts as a general diffusion porin. The present study employed planar black lipid membrane (BLM) reconstitution to demonstrate the single-channel conductance of the trimeric BpsOmp38. Hightime resolution BLM measurements displayed ion current blockages of each antimicrobial agent in a concentration-dependent manner with the translocation on-rate (kon) following the order: norfloxacin>> ertapenem > ceftazidime > cefepime > imipenem > meropenem >> penicillin G. The dwell time of a selected antimicrobial agent (ertapenem) decayed exponentially with increasing temperature. The energy barrier for the ertapenem binding to the affinity site inside the BpsOmp38 channel was estimated from the Arrhenius plot to be 12 kT and for the ertapenem release to be 13 kT at +100 mV. Topology prediction suggested that Tyr-119, located in the middle of the pore, may act as a pore-constricting residue that controls the permeability of hydrophilic species through BpsOmp38. Antibiotic susceptibility assay showed that the Omp-deficient E. coli expressing BpsOmp38Y119A and Y119F mutants were more susceptible to meropenem and ceftazidime, but less susceptible to doripenem when compared with the susceptibility of E. coli expressing BpsOmp38 wild-type. Liposome-swelling assay suggested that the permeability rate of neutral sugars through the BpsOmp38 variants increased as the molecular weight of the sugar decreased. However, the permeation of antibiotics through the BpsOmp38 porins showed modest correlation between the molecular weight and the diffusion rates. The result suggested that not only the pore diameter, but the interactions between the antibiotic and the residues lining the pore interior also play an important role in the molecular uptake through BpsOmp38.