Diagnosis of lung cancer requires radiologists to review every lung nodule in CT images. Such a process can be very time-consuming, and the accuracy is affected by many factors, such as experience of radiologists and available diagnosis time. To address this problem, we proposed to develop a deep learning-based system to automatically classify benign and malignant lung nodules.The proposed method automatically determines benignity or malignancy given the 3D CT image patch of a lung nodule to assist diagnosis process. Motivated by the fact that real structure among data is often embedded on a low-dimensional manifold, we developed a novel manifold regularized classification deep neural network (MRC-DNN) to perform classification directly based on the manifold representation of lung nodule images. The concise manifold representation revealing important data structure is expected to benefit the classification, while the manifold regularization enforces strong, but natural constraints on network training, preventing over-fitting.The proposed method achieves accurate manifold learning with reconstruction error of ~ 30 HU on real lung nodule CT image data. In addition, the classification accuracy on testing data is 0.90 with sensitivity of 0.81 and specificity of 0.95, which outperforms state-of-the-art deep learning methods.The proposed MRC-DNN facilitates an accurate manifold learning approach for lung nodule classification based on 3D CT images. More importantly, MRC-DNN suggests a new and effective idea of enforcing regularization for network training, possessing the potential impact to a board range of applications.