Abstract Probiotic lactobacilli colonize mucosae and benefit human health by preventing infections, reducing cancer risk, and improving metabolism. Among the lactobacilli, Lactobacillus plantarum is one of the most widely studied probiotics. Lactobacilli use surface molecules called adhesins to form physical associations with host tissues, and serine-rich repeat proteins (SRRPs) are one specific class of adhesin used by probiotics and pathogens alike within in the Lactobacillaceae. A key gap in our knowledge is how these proteins evolve specificity for their colonization site within the host. This proposal investigates the hypothesis that cell-cell competition inside the host creates an evolutionary selection pressure on SRRP genes to maximize bacterial cell fitness inside the host. We investigate the first SRRP genes discovered in L. plantarum, which we found to be necessary for colonization of a specific physical niche in the host digestive tract. The proposal will examine evolution and specialization of the SRRP genes as well as the functions of specific subdomains of the protein for their role in binding host mucus vs forming a biofilm, vs promoting bacterial growth within the niche by enzymatically liberating host glycans. We will use in vivo assays to connect the molecular properties of the SRRPs to cellular fitness in the host. These assays include imaging the bacteria at single cell resolution inside the guts of living animals to visualize the cell-cell competition directly. Success in this work could develop an unparalleled model system to study the evolution of colonization specificity. Due to the wide conservation of SRRPs in binding host mucus, this model may reveal conserved genetic mechanisms that can be applied to develop better probiotics for humans and combat mucosal pathogens.