Abstract: Objective: This study focused on the Lactiplantibacillus plantarum WCFS1 strain to investigate the structure–function relationship of its exopolysaccharide (EPS) biosynthesis gene clusters (cps), thereby laying the foundation for further research into strain-specific characteristics of lactic acid bacteria EPS. Methods: Mutant strains WCFS1∆cps1-3 and WCFS1∆cps4 were constructed using Cre-loxP homologous recombination and CRISPR/Cas9 techniques. The growth characteristics, EPS yield, and monosaccharide composition of the mutant strains were analyzed using the wild-type strain as a control. Transcriptomic analysis was performed to examine the functional roles of differentially expressed genes in the WCFS1∆cps1-3 mutant under three distinct culture conditions. Results: The WCFS1∆cps4 mutant exhibited a greater reduction in EPS yield, dropping to 62.2% of the wild-type level. This was accompanied by slow growth, with the time to reach the stationary phase delayed from 12 h to 32 h. However, biomass remained unaffected. The monosaccharide composition of EPS showed significant changes in the WCFS1∆cps1-3 mutant, with mannose content increasing markedly to 32.68% and galactose content decreasing significantly to 3.83%, while rhamnose was undetectable. The number of significantly differentially expressed genes in the WCFS1∆cps1-3 mutant compared to that in the wild-type strain under varying culture conditions accounted for only 5.1% (upregulated) and 6.8% (downregulated) of those observed under MRS culture conditions, with GO and KEGG analyses revealing substantial functional differences. Conclusion: The structure–function relationships of the cps clusters in the WCFS1 strain varied. The highly conserved cps4 cluster may regulate EPS yield to ensure normal bacterial growth. However, the cps1-3 cluster likely modulates the monosaccharide composition of EPS, conferring greater environmental adaptability to the strain.