Abstract Brain extraction is a critical pre-processing step in brain magnetic resonance imaging (MRI) analytical pipelines. In rodents, this is often achieved by manually editing brain masks slice-by-slice, a time-consuming task where workloads increase with higher spatial resolution datasets. We recently demonstrated successful automatic brain extraction via a deep-learning-based framework, U-Net, using 2D convolutions. However, such an approach cannot make use of the rich 3D spatial-context information from volumetric MRI data. In this study, we advanced our previously proposed U-Net architecture by replacing all 2D operations with their 3D counterparts and created a 3D U-Net framework. We trained and validated our model using a recently released CAMRI rat brain database acquired at isotropic spatial resolution, including T2-weighted turbo-spin-echo structural MRI and T2*-weighted echo-planar-imaging functional MRI. The performance of our 3D U-Net model was compared with existing rodent brain extraction tools, including Rapid Automatic Tissue Segmentation (RATS), Pulse-Coupled Neural Network (PCNN), SHape descriptor selected External Regions after Morphologically filtering (SHERM), and our previously proposed 2D U-Net model. 3D U-Net demonstrated superior performance in Dice, Jaccard, Hausdorff distance, and sensitivity. Additionally, we demonstrated the reliability of 3D U-Net under various noise levels, evaluated the optimal training sample sizes, and disseminated all source codes publicly, with a hope that this approach will benefit rodent MRI research community. Significant methodological contribution We proposed a deep-learning-based framework to automatically identify the rodent brain boundaries in MRI. With a fully 3D convolutional network model, 3D U-Net, our proposed method demonstrated improved performance compared to current automatic brain extraction methods, as shown in several qualitative metrics (Dice, Jaccard, PPV, SEN, and Hausdorff). We trust that this tool will avoid human bias and streamline pre-processing steps during 3D high resolution rodent brain MRI data analysis. The software developed herein has been disseminated freely to the community.