Burkholderia pseudomallei, the causative agent of the high-mortality disease melioidosis, is a Gram-negative saprophytic bacterium that is naturally resistant to most antibiotics. There is no vaccine for melioidosis, with treatment reliant on biphasic and prolonged antibiotic administration. The primary treatment option is ceftazidime, and for severe cases, the carbapenem drug meropenem. Intrinsic B. pseudomallei resistance towards meropenem has not yet been documented; however, resistance could conceivably develop over the course of infection, leading to prolonged sepsis and difficulties in infection eradication, which could be fatal. Here, we document twelve melioidosis cases whereby B. pseudomallei isolates developed decreased susceptibility towards meropenem during treatment. Minimum inhibitory concentrations for the initial strains were 0.75-1 µg/mL, but increased to 3-8 µg/mL after antibiotic administration. Using whole-genome sequencing, we identified multiple mutations affecting resistance-nodulation-division (RND) multidrug efflux pump regulators that were associated with decreased meropenem susceptibility, with all mechanisms related to the loss of efflux pump regulation. Our study sheds light on the role of RND efflux pumps in decreasing meropenem susceptibility in B. pseudomallei, and documents a concerning example of acquired resistance towards one of the last and most critical treatment options for melioidosis.