Heat and enzyme treatments were used to increase the prebiotic oligosaccharides from palm kernel expeller (PKE), and the prebiotic efficacy of three types of PKE-extracts, namely raw PKE-extract (PKERAW), enzyme-treated PKE-extract (PKEENZ), and steam + enzyme-treated PKE-extract (SPKEENZ) was evaluated in vitro using three strains of Lactobacillus (L. brevis I 218, L. salivarius I 24 and L. gallinarum I 16), and in vivo using Sprague-Dawley rats as an animal model. Results of the in vitro study showed that the PKE-extracts were able to support the growth of Lactobacillus sp. strains. However, their growth varied significantly among strains and PKE-extracts (P<0.05), with L. brevis I 218 recording the highest growth compared to the other two strains, and the highest growth in the steam plus enzyme (SPKEENZ) extract. Results of the in vivo study reaffirmed that all the PKE-extracts tested can support growth of beneficial bacteria (Lactobacillus and Bifidobacterium), but only SPKEENZ treatment group had significantly higher Lactobacillus and Bifidobacterium counts and lower population of E. coli compared to the control. It was demonstrated that PKE is a potential source of prebiotic, which may be used to effectively improve host health and wellbeing by modulating the host gut microflora, and by proper pre-treatment, the release of prebiotic oligosaccharides from PKE can be enhanced.