Abnormal N-glycosylation of uromodulin (Umod, also known as Tamm-Horsfall protein) has been implicated in the pathogenesis of various urinary system diseases. However, the site-specific N-glycosylation patterns of urinary Umod in healthy controls (HCs) versus those with kidney stones (KSs) have yet to be detailed. In this study, we isolated and purified Umod from the urine of 24 patients with calcium oxalate (CaOx) KSs and 24 HCs using a refined diatomaceous earth adsorption method. After digestion with trypsin and Glu-C, intact N-glycopeptides (IGPs) were enriched via hydrophilic interaction liquid chromatography (HILIC) and analyzed via EThcD-sceHCD-MS/MS. Data processing was conducted using Byonic and PANDA software. A total of 700 IGPs, 8 N-glycosites, and 145 N-glycans were identified, marking the most extensive identification of N-glycosylation in Umod to date. Through quantitative and site-specific N-glycosylation analyses, the study found 39 IGPs to be up-regulated (p<0.05, FC>1.5) and 60 down-regulated IGPs (p<0.05, FC<0.67) in KS groups. Additionally, the analysis of N-glycan composition and site-specific N-glycosylation highlighted KS-specific N-glycosylation modifications. Overall, these results suggest that variations in Umod N-glycosylation may critically contribute to the formation of CaOx kidney stones by modulating its function.