Preliminary research identified three polysaccharide fractions, namely— VL8-EPS30, VL8-EPS50, and VL8-EPS70,— isolated from Lactobacillus. paracasei EPS using graded ethanol precipitation. These fractions varied in yield, composition (Total sugar, protein, and uronic acid), monosaccharide profile, molecular weight, stability, and morphology. Despite these differences, all promoted RAW 264.7 cell proliferation. Among them, VL8-EPS50 exhibited the highest total sugar and uronic acid content, along with branched and folded structures, enhancing its macrophage activation, phagocytic activity, and nitric oxide (NO) secretion. However, its immunomodulatory mechanisms and effects on gut microbiota remain unclear. To investigate these mechanisms, we administered VL8-EPS50 at different doses to cyclophosphamide (CTX)-induced immunosuppressed mice. VL8-EPS50 significantly enhanced splenic lymphocyte proliferation and increased immunoglobulin and serum cytokine secretion. Additionally, it alleviated CTX-induced gut microbiota dysbiosis by increasing microbial diversity and richness, promoting beneficial bacteria (Lactobacillus), reducing pathogenic genera (Alistipes), and enhancing short-chain fatty acid production. These findings establish VL8-EPS50 as a promising immunomodulator that influences immune function through gut microbiota regulation. This research supports its potential application in developing functional dairy products with immunological benefits.