Validation of a new single breath-hold, three-dimensional, cine balanced steady-state free precession (3D cine bSSFP) cardiac magnetic resonance (CMR) sequence for left ventricular function. CMR examinations were performed on fifteen patients and three healthy volunteers on a clinical 1.5T scanner using a two-dimensional (2D) cine balanced SSFP CMR sequence (2D cine bSSFP) followed by an investigational 3D cine bSSFP pulse sequence acquired within a single breath hold. Left ventricular end diastolic volume (LVEDV), end systolic volume (LVESV), ejection fraction (LVEF), and myocardial mass were independently segmented on a workstation by two experienced radiologists. Blood pool to myocardial contrast was evaluated in consensus using a Likert scale. Bland–Altman analysis was used to compare these quantitative and nominal measurements for the two sequences. The average acquisition time was significantly shorter for the 3D cine bSSFP than for 2D cine bSSFP (0.36 ± 0.03 vs. 8.5 ± 2.3 min) p = 0.0002. Bland–Altman analyses [bias and (limits of agreement)] of the data derived from these two methods revealed that the LVEF 0.9 % (−4.7, 6.4), LVEDV 4.9 ml (−23.0, 32.8), LVESV −0.2 ml (−22.4, 21.9), and myocardial mass −0.4 g (−23.8, 23.0) were not significantly different. There was excellent intraclass correlation for intra-observer variability (0.981, 0.989, 0.997, 0.985) and inter-observer variability (0.903, 0.954, 0.970, 0.842) for LVEF, LVEDV, LVESV, and myocardial mass respectively. 3D cine bSSFP allows for accurate single breath-hold volumetric cine CMR which enables substantial improvements in scanner time efficiency without sacrificing diagnostic accuracy.