<p><i>Lactobacillaceae</i> are a large, diverse family of Gram-positive lactic acid-producing bacteria. As gut microbiota residents in many mammals, these bacteria are beneficial for health and frequently used as probiotics. <i>Lactobacillaceae</i> abundance in the gastrointestinal tract has been correlated with gastrointestinal pathologies and infection. Microbiota residents must compete for nutrients, including essential metal ions like calcium, zinc, and iron. Recent animal and human studies have revealed that dietary calcium can positively influence the diversity of the gut microbiota and abundance of intestinal <i>Lactobacillaceae</i> species, but the underlying molecular mechanisms remain poorly understood. Here, we investigated the impacts of calcium on the growth and biofilm formation of two distinct <i>Lactobacillaceae</i> species found in the gut microbiota, <i>Lactobacillus acidophilus</i> ATCC 4356 and <i>Lactiplantibacillus plantarum</i> ATCC 14917. We found that calcium ions differentially affect both growth and biofilm formation of these species. In general, calcium supplementation promotes the growth of both species, albeit with some variations in the extent to which different growth parameters were impacted. Calcium ions strongly induce biofilm formation of <i>L. acidophilus</i> ATCC 4356 but not <i>L. plantarum</i> ATCC 14917. Based on bioinformatic analyses and experimental chelator studies, we hypothesize that surface proteins specific to <i>L. acidophilus</i> ATCC 4356, like S-layer proteins, are responsible for Ca²⁺-induced biofilm formation. The ability of bacteria to form biofilms has been linked with their ability to colonize in the gut microbiota. This work shows how metal ions like Ca²⁺ may be important not just as nutrients for bacteria growth, but also for their ability to facilitate cell-cell interactions and possibly colonization in the gut microbiota.</p>