Backgrounds: Previous studies have demonstrated that excretion of urinary extracellular vesicles (EVs) from different nephron segments differs between kidney stone formers and non-stone formers (NSFs), and could reflect pathogenic mechanisms of urinary stone disease. In this study we quantified selected populations of specific urinary EVs carrying protein markers of immune cells and calcium/phosphorus physiology in calcium stone formers (CSFs) compared to non-stone formers (NSFs). Methods Incident CSFs (n = 24) and age- and sex- matched NSFs (n = 21) were studied. Clinical data were abstracted and biobanked cell-free urine samples were used to quantify specific urinary EV populations. EVs carrying proteins related to renal calcium/phosphorus physiology (phosphorus transporters (PiT1 and PiT2), Klotho, and fibroblast growth factor 23 (FGF23)); markers associated with EV generation (anoctamin-4 (ANO4) and Huntington interacting protein 1 (HIP1)), and markers shed from activated immune cells were quantified by standardized and published method of digital flow cytometry. Results The urine pH of CSFs was lower than NSFs (P < 0.05), whereas urine excretion of calcium, phosphorus, and calcium oxalate and uric acid supersaturation (SS) were significantly higher in CSFs compared to NSFs (P < 0.05). Urinary excretion of EVs with markers of total leukocytes (CD45), neutrophils (CD15), macrophages (CD68), Klotho, FGF23, PiT1, PiT2, and ANO4 were each markedly lower in CSFs than NSFs (P < 0.05) whereas excretion of those with markers of monocytes (CD14), T-Lymphocytes (CD3), B-Lymphocytes (CD19), plasma cells (CD138 plus CD319 positive ) were not different between the groups. Urinary excretion of EVs expressing PiT1 and PiT2 negatively (P < 0.05) correlated with urinary phosphorus excretion whereas excretion of EVs expressing FGF23 correlated negatively (P < 0.05) with both urinary calcium and phosphorus excretion. Conclusions Urinary excretion of EVs derived from specific types of activated immune cells and EVs with proteins related to calcium/phosphorus regulation were different between CSFs and NSFs. Thus, further validation of these and other populations of urinary EVs could identify biomarkers that elucidate novel pathogenic mechanisms of calcium stone formation in specific subsets of patients.