<p>The gut microbiota (GM) is increasingly acknowledged for its regulatory role in host immune responses and its influence on various systemic diseases. Nevertheless, whether GM modulates kidney stone disease (KSD) through immune-related mechanisms remains uncertain. To investigate these associations, we conducted Mendelian randomization (MR) analyses in two sequential steps, employing summary-level data sourced from comprehensive genome-wide association studies (GWAS). Specifically, the potential causal influence of 430 GM features on 731 immune-cell-related traits, and subsequently their effects on KSD susceptibility, was assessed. The primary analysis utilized the inverse variance weighted (IVW) method, alongside complementary approaches including MR-Egger regression, weighted median analysis, and various sensitivity evaluations. Moreover, a mediation MR analysis was conducted to determine if specific immune cell subsets mediated relationships between GM and KSD. Our analysis revealed significant causal associations between 15 GM taxa and KSD. Protective effects were observed for genera such as Prevotella, Phascolarctobacterium, and Ruminococcaceae, whereas elevated risks of KSD were associated with Bacteroides, Clostridiales, and Subdoligranulum. Furthermore, 35 immune-cell-related phenotypes displayed causal links to KSD risk. Importantly, CD28 expression on CD39 + CD4 + T cells was identified as a mediator in the protective pathway from Prevotella to KSD, with a mediation effect estimated at –9.019% (<i>P</i> = 0.044). No significant directional pleiotropy or heterogeneity was observed. These findings provide novel causal evidence supporting the existence of a gut–immune–kidney axis, where certain gut microbes influence KSD susceptibility via immune mechanisms. This may guide the development of microbiota- or immune-targeted strategies for the prevention of KSD.</p>