Objectives: To evaluate the correlation between vitamin K-dependent gamma-glutamyl carboxylase (GGCX) and the formation of calcium oxalate urolithiasis (COU). Methods: Renal cortex samples were obtained from patients undergoing nephrectomy and divided into 3 groups: (1) urolithiasic group (n = 44); (2) hydronephrosis-without-stone group (control group A, n = 6), and (3) normal control group (control group B, n = 21). Immunohistochemical technique was established to study the location of GGCX in renal tissues. The carboxylase activity was detected by the isotope-labeled carboxylatic reaction in vitro. We also quantified GGCX mRNA levels using TaqMan real-time reverse-transcription PCR, and protein was detected by Western blot. Results: GGCX was located in the cytoplasm of renal tubular epithelial cells and the activity of GGCX in the urolithiasic group was significantly decreased compared with that of controls (p < 0.05). GGCX mRNA in the urolithiasic group was lower than that in the normal control group, which was on average 7.86-fold underexpressed in the urolithiasic group compared to the normal control group. Protein expression of GGCX in the urolithiasic group (27.64 ± 0.29) was weaker than that in the other 2 control groups (control group A 55.22 ± 0.36, control group B 53.78 ± 0.33), p < 0.05. Conclusions: The activity and expression of GGCX are decreased in renal tissues of patients with COU.