The purpose of this study was to investigate the effects of cardiac pulsation in diffusion tensor imaging (DTI) of the rat brain. DTI data were acquired either with or without different cardiac gating delays. For each case, two sets of identical DTI data were acquired for a bootstrap analysis to measure the uncertainty in estimating mean diffusivity (MD), fractional anisotropy (FA) and the primary eigenvector direction. The 95% confidence interval of the primary eigenvectors was substantially reduced (21-25%) when cardiac gating with triggering delay of 70 ms (∼half of R-R peak duration) was used in comparison to studies without gating or when gating with a triggering delay of 0 ms was used. Standard deviations of MD and FA estimates were also reduced by 12-26% and 13-24%, respectively. For voxels with mean FA values larger than 0.15 and smaller than 0.95, the decrease in CI and standard deviations of MD and FA by cardiac gating with triggering delay of 70 ms were significant (p < 0.05). These results demonstrate the importance of cardiac gating in acquisition of in vivo high resolution DTI data.