Background— The aim of this study is to explore the differential effect of 3-dimensional color Doppler echocardiography for the quantification of mitral regurgitation (MR). Two-dimensional color Doppler echocardiography–based MR quantification has well-documented limitations. Methods and Results— We consecutively enrolled 221 patients with MR. Adequate image quality was obtained by 2D- and 3D-color Doppler echocardiography in 211 (95.5%) patients. The quantitative differences between the MR volumes obtained by 2D- and 3D-proximal isovelocity surface area (PISA) were analyzed in various MR subgroups. In the validation cohort (n=52), MR volume obtained by 3D-PISA showed a better agreement with phase-contrast cardiac MRI than 2D-PISA ( r =0.97 versus 0.84). In all 211 patients, 2D-PISA underestimated the MR volume when compared with 3D-PISA (52.4±19.6 versus 59.5±25.6 mL; P =0.005). A total of 33.3% with severe MR based on 3D-PISA were incorrectly assessed by 2D-PISA as having nonsevere MR. In the subgroup analysis, the MR severity (odds ratio, 6.96; 95% confidence interval, 3.04–15.94; P <0.001) and having an asymmetrical orifice (odds ratio, 11.48; 95% confidence interval, 3.72–35.4; P <0.001), and an eccentric jet (odds ratio, 3.82; 95% confidence interval, 1.27–11.48; P =0.017) were predictors of significant difference in MR volume (>15 mL) between 2D- and 3D-PISA methods. Conclusions— Quantification of MR by 3D-PISA method is clinically feasible and more accurate than the current 2D-PISA method. MR quantification by 2D-PISA significantly underestimated MR volume with severe, eccentric MR with an asymmetrical orifice. This article demonstrates that 3D-color Doppler echocardiography could be used as a valuable tool to confirm treatment strategy in patients with significant MR.