The gut microbiota (GM) are intimately correlated with the formation and progression of nephrolithiasis. However, the specific causal relationships and mechanisms between the GM and nephrolithiasis remain unclear. This study aims to explore the molecular mechanisms of key genes related to the GM in nephrolithiasis. The nephrolithiasis genome-wide association studies (GWAS) data (ukb-b-18372), GM data, GSE73680, GSE117518 and GSE231569 were all derived from public databases. Firstly, the GMs causally related to nephrolithiasis were identified through Mendelian randomization (MR), sensitivity analysis and Steiger test. Subsequently, key genes were determined through differential expression analysis and expression analysis. In addition, functional enrichment, construction of molecular regulatory networks, drug prediction and molecular docking were carried out, and key cells were identified through single-cell analysis. It was confirmed by the MR analysis that 35 GMs had a causal relationship with nephrolithiasis, and PALLD and R3HDM1 were identified as key genes. Enrichment analysis indicated that PALLD and R3HDM1 were co-enriched in multiple pathways, such as oxidative phosphorylation. The miRNA-mRNA network revealed that 9 miRNAs could act on both PALLD and R3HDM1 simultaneously, such as hsa-miR-1256. Drug prediction analysis found that drugs like benzo(a)pyrene could co-target PALLD and R3HDM1, and PALLD had the strongest binding ability with ICG-001, with a binding energy of -9.5 kcal/mol. Finally, fibroblasts (FIBs) and vascular smooth muscle cells/pericytes (VSM/Ps) were determined as key cells, and the expression of PALLD exhibited nonlinear change characteristics during the differentiation process of FIBs and VSM/Ps. PALLD and R3HDM1 were identified as GM-related key genes in nephrolithiasis, providing a reference for exploring the pathogenesis of nephrolithiasis.