Lung lobe segmentation in chest CT has been used for the analysis of lung functions and surgical planning. However, accurate lobe segmentation is difficult as 80% of patients have incomplete and/or fake fissures. Furthermore, lung diseases such as chronic obstructive pulmonary disease (COPD) can increase the difficulty of differentiating the lobar fissures. Lobar fissures have similar intensities to those of the vessels and airway wall, which could lead to segmentation error in automated segmentation. In this study, a fully automated lung lobe segmentation method with 3D U-Net was developed and validated with internal and external datasets. The volumetric chest CT scans of 196 normal and mild-to-moderate COPD patients from three centers were obtained. Each scan was segmented using a conventional image processing method and manually corrected by an expert thoracic radiologist to create gold standards. The lobe regions in the CT images were then segmented using a 3D U-Net architecture with a deep convolutional neural network (CNN) using separate training, validation, and test datasets. In addition, 40 independent external CT images were used to evaluate the model. The segmentation results for both the conventional and deep learning methods were compared quantitatively to the gold standards using four accuracy metrics including the Dice similarity coefficient (DSC), Jaccard similarity coefficient (JSC), mean surface distance (MSD), and Hausdorff surface distance (HSD). In internal validation, the segmentation method achieved high accuracy for the DSC, JSC, MSD, and HSD (0.97 ± 0.02, 0.94 ± 0.03, 0.69 ± 0.36, and 17.12 ± 11.07, respectively). In external validation, high accuracy was also obtained for the DSC, JSC, MSD, and HSD (0.96 ± 0.02, 0.92 ± 0.04, 1.31 ± 0.56, and 27.89 ± 7.50, respectively). This method took 6.49 ± 1.19 s and 8.61 ± 1.08 s for lobe segmentation of the left and right lungs, respectively. Although various automatic lung lobe segmentation methods have been developed, it is difficult to develop a robust segmentation method. However, the deep learning-based 3D U-Net method showed reasonable segmentation accuracy and computational time. In addition, this method could be adapted and applied to severe lung diseases in a clinical workflow.