Osteoarthritis (OA) is the most common form of arthritis and can often occur in the knee. While convolutional neural networks (CNNs) have been widely used to study medical images, the application of a 3-dimensional (3D) CNN in knee OA diagnosis is limited. This study utilizes a 3D CNN model to analyze sequences of knee magnetic resonance (MR) images to perform knee OA classification. An advantage of using 3D CNNs is the ability to analyze the whole sequence of 3D MR images as a single unit as opposed to a traditional 2D CNN, which examines one image at a time. Therefore, 3D features could be extracted from adjacent slices, which may not be detectable from a single 2D image. The input data for each knee were a sequence of double-echo steady-state (DESS) MR images, and each knee was labeled by the Kellgren and Lawrence (KL) grade of severity at levels 0–4. In addition to the 5-category KL grade classification, we further examined a 2-category classification that distinguishes non-OA (KL ≤ 1) from OA (KL ≥ 2) knees. Clinically, diagnosing a patient with knee OA is the ultimate goal of assigning a KL grade. On a dataset with 1100 knees, the 3D CNN model that classifies knees with and without OA achieved an accuracy of 86.5% on the validation set and 83.0% on the testing set. We further conducted a comparative study between MRI and X-ray. Compared with a CNN model using X-ray images trained from the same group of patients, the proposed 3D model with MR images achieved higher accuracy in both the 5-category classification (54.0% vs. 50.0%) and the 2-category classification (83.0% vs. 77.0%). The result indicates that MRI, with the application of a 3D CNN model, has greater potential to improve diagnosis accuracy for knee OA clinically than the currently used X-ray methods.