Gliomas are rapidly progressive, neurologically devastating, largely fatal brain tumors. Magnetic resonance imaging (MRI) is a widely used technique employed in the diagnosis and management of gliomas in clinical practice. MRI is also the standard imaging modality used to delineate the brain tumor target as part of treatment planning for the administration of radiation therapy. Despite more than 20 yr of research and development, computational brain tumor segmentation in MRI images remains a challenging task. We are presenting a novel method of automatic image segmentation based on holistically nested neural networks that could be employed for brain tumor segmentation of MRI images. Two preprocessing techniques were applied to MRI images. The N4ITK method was employed for correction of bias field distortion. A novel landmark-based intensity normalization method was developed so that tissue types have a similar intensity scale in images of different subjects for the same MRI protocol. The holistically nested neural networks (HNN), which extend from the convolutional neural networks (CNN) with a deep supervision through an additional weighted-fusion output layer, was trained to learn the multiscale and multilevel hierarchical appearance representation of the brain tumor in MRI images and was subsequently applied to produce a prediction map of the brain tumor on test images. Finally, the brain tumor was obtained through an optimum thresholding on the prediction map. The proposed method was evaluated on both the Multimodal Brain Tumor Image Segmentation (BRATS) Benchmark 2013 training datasets, and clinical data from our institute. A dice similarity coefficient (DSC) and sensitivity of 0.78 and 0.81 were achieved on 20 BRATS 2013 training datasets with high-grade gliomas (HGG), based on a two-fold cross-validation. The HNN model built on the BRATS 2013 training data was applied to ten clinical datasets with HGG from a locally developed database. DSC and sensitivity of 0.83 and 0.85 were achieved. A quantitative comparison indicated that the proposed method outperforms the popular fully convolutional network (FCN) method. In terms of efficiency, the proposed method took around 10 h for training with 50,000 iterations, and approximately 30 s for testing of a typical MRI image in the BRATS 2013 dataset with a size of 160 × 216 × 176, using a DELL PRECISION workstation T7400, with an NVIDIA Tesla K20c GPU. An effective brain tumor segmentation method for MRI images based on a HNN has been developed. The high level of accuracy and efficiency make this method practical in brain tumor segmentation. It may play a crucial role in both brain tumor diagnostic analysis and in the treatment planning of radiation therapy.