Abstract <p><i>Lacticaseibacillus rhamnosus</i> strain GG (LGG) is a broadly used probiotic with several unique features that help it provide beneficial effects to its host. Key among the probiotic features of LGG is the production of bioactive metabolites and secreted proteins such as p40 and p75. The ability of LGG to persist in the gastrointestinal tract depends primarily on its ability to adhere to the gut mucosa via the generation of adhesion pili. While LGG is already used as a probiotic, potential still exists for optimization of the metabolic state of LGG to further enhance its probiotic capacity. Here, we evaluated the ability of whey protein isolate to enhance the cell growth and probiotic effects of LGG. RNA sequencing and untargeted metabolomics revealed that WPI supplementation enhanced LGG growth and metabolic activity and increased the transcription of genes for the production of adhesion pili, key secreted proteins, and beneficial metabolites. These results indicate that whey protein is a viable supplement option for use with LGG and may help to boost the probiotic activity and growth of LGG within the gastrointestinal tract.</p> Key points <p>• <i>Whey protein isolate supplementation increases Lacticaseibacillus rhamnosus GG growth.</i></p> <p>• <i>Transcription of genes for probiotic features is amplified by the addition of WPI.</i></p> <p>• <i>Transcriptomics and metabolomics suggest the protein produces the beneficial effects.</i></p>