Burkholderia pseudomallei causes the disease melioidosis in humans and is classified as a category B select agent. Research utilizing this pathogen is highly regulated in the United States, and even basic studies must be conducted in biosafety level 3 (BSL-3) facilities. There is currently no attenuated B. pseudomallei strain available that is excluded from select-agent regulations and can be safely handled at BSL-2 facilities. To address this need, we created Bp82 and Bp190, which are DeltapurM derivatives of B. pseudomallei strains 1026b and K96243 that are deficient in adenine and thiamine biosynthesis but replication competent in vitro in rich medium. A series of animal challenge studies was conducted to ensure that these strains were fully attenuated. Whereas the parental strains 1026b and K96243 and the complemented mutants Bp410 and Bp454 were virulent in BALB/c mice following intranasal inoculation, the DeltapurM mutants Bp82 and Bp190 were avirulent even when they were administered at doses 4 logs higher than the doses used for the parental strains. Animals challenged with high doses of the DeltapurM mutants rapidly cleared the bacterium from tissues (lung, liver, and spleen) and remained free of culturable bacteria for the duration of the experiments (up to 60 days postinfection). Moreover, highly susceptible 129/SvEv mice and immune incompetent mice (IFN-gamma-/-, SCID) were resistant to challenges with DeltapurM mutant Bp82. This strain was also avirulent in the Syrian hamster challenge model. We concluded that DeltapurM mutant Bp82 is fully attenuated and safe for use under BSL-2 laboratory conditions and thus is a candidate for exclusion from the select-agent list.