The mechanisms of bacterial adhesion to human cells involve several complex reactions and activation of genes and proteins. It has been reported that the food components in dairy matrices, such as sugar or salt, can decrease bacterial adhesion to Caco-2 cells. However, it has not been evaluated whether the bacteria grown in media supplemented with milk phospholipids (MPL) can increase or decrease the adhesion of these cells. The objective of this work was to evaluate the effects of MPL on the kinetic growth of lactic acid bacteria (LAB) and their functional characteristics as probiotics, expression of surface protein genes, and adherence to Caco-2 cells. Seven LAB strains isolated from various dairy products were characterized. Five of the tested LAB strains were able to grow in a chemically defined medium supplemented with MPL. Lactobacillus reuteri OSU-PECh-48 showed the highest growth rate and the greatest optical density. All of the strains tested showed tolerance to acidic conditions at pH 3.0 and to bile salts at 0.5 and 1% concentrations. Auto-aggregation and cell surface hydrophobicity ability were evaluated, with nonsignificant differences between the strains grown in MPL and without MPL. Gene expression of 6 surface proteins was evaluated in the presence or absence of MPL. Pediococcus acidilactici OSU-PECh-L and OSU-PECh-48 were the strains with highest relative expression of 5 of the 6 genes evaluated. Lactobacillus paracasei OSU-PECh-BA was the strain with the lowest level of expression of surface protein genes. Most of the bacteria tested had increased adhesion to Caco-2 cells after growth in MPL. The bacteria with the highest degrees of adhesion observed were Lactobacillus paracasei OSU-PECh-3B, Pediococcus acidilactici OSU-PECh-L, and Lactobacillus reuteri OSU-PECh-48. The genes Cnb and EF-Tu increased in expression in the presence of MPL in most of the LAB tested. The results obtained in this work demonstrate the high potential of these LAB strains for use as starters or beneficial cultures in fermentation of not only dairy products but also other food fermentation processes, with promising ability to increase residence time in the gut, modify the microbiome, and improve human health.