Lactobacillus strains are known to produce exopolysaccharides (EPS) with recognized health benefits (i.e. prebiotic and immunomodulation) but production is limited by low yields. Co-culture has been shown to improve metabolite productivity, particularly bacteriocins and EPS. Although lactic acid bacteria (LAB) and yeasts are found in several fermented products, the molecular mechanisms linked to the microbial interactions and their influences on EPS biosynthesis are unclear. The aim of the present study was to investigate the effect of co-culture on EPS production by three Lactobacillus rhamnosus strains (ATCC 9595, R0011, and RW-9595M) in association with Saccharomyces cerevisiae. Fermentation, in both mono and co-culture, was carried out and the expression of key LAB genes was monitored. After 48 h, results revealed that EPS production was enhanced by 39%, 49%, and 42% in co-culture for R0011, ATCC 9595, and RW-9595M, respectively. Each strain showed distinctive gene expression profiles. For a higher EPS production, higher EPS operon expression levels were observed for RW-9595M in co-culture. The construction of gene co-expression networks revealed common correlations between the expression of genes related to the EPS operons, sugar metabolism, and stress during EPS production and microbial growth for the three strains. Our findings provide insight into the positive influence of inter-kingdom interactions in stimulating EPS biosynthesis, representing progress toward the development of a bio-ingredient with broad industrial applications.