The crystallites in urine are related closely with the formation of urolithiasis. In the present paper the composition, morphology and Zeta potential of crystallites of twenty calcium oxalate stone formers were comparatively studied using X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, nanoparticle size analyzer, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results showed that calcium oxalate calculi usually coexisted with a little of uric acid, calcium phosphate, and magnesium ammonium phosphate. By contrast, the compositions of urine crystallites of the patients with calcium oxalate calculi were mainly uric acid, phosphate, calcium oxalate and so on. Most of them had sharp angularity with a particle size distribution ranging from tens of nanometers to tens of microns; and obvious aggregation was observed. The negative value of Zeta potential of urine crystallites in the twenty stone formers (average value -5.92 mV) was less than that in the twenty normal subjects (-12.9 mV). However, there was no obvious difference in the urine pH between stone formers (average pH 6.03) and normal subjects (average pH 5.92). The study on the relationship between urine crystallites and urinary calculi components will be helpful for finding out the causes of urolithiasis and providing an important basis for the scientific prevention methods and reasonable treatments in clinic.