Formation of potential microbial inhibitors during pretreatment and hydrolysis of lignocellulosic biomass has long been identified as one of the key factors that affect bioconversion of biomass to biofuels and chemicals. In this study, a direct adaptation process was carried out with dried distillers' grains and solubles (DDGS) hydrolysates when employing the hyper-butanol producing Clostridium beijerinckii BA101. The results demonstrated that the adapted cells were able to adjust to the inhibitory environment in a much shorter time and produce approximately the same amount of ABE as the control fermentation employing unadapted cells. Further iterative adaptation processes were applied and the results demonstrated that further tolerance was obtained. Challenge of the microorganism using the inhibitors present in the DDGS hydrolysate is believed to have increased the ability of C. beijerinckii to tolerate the presence of these inhibitors and resulted in significantly shorter lag phase. Experiments for elucidation of the molecular mechanism underlying the adaptation process are underway. The promising results from this study can provide further encouragement for the study of tolerance of the solventogenic clostridia to inhibitors and development of inhibitor-tolerant strains.