Osteosarcoma is a common type of bone tumor, particularly prevalent in children and adolescents between the ages of 5 and 25 who are experiencing growth spurts during puberty. Manual delineation of tumor regions in MRI images can be laborious and time-consuming, and results may be subjective and difficult to replicate. Therefore, a convolutional neural network (CNN) was developed to automatically segment osteosarcoma cancerous cells in three types of MRI images. The study consisted of five main stages. First, 3692 DICOM format MRI images were acquired from 46 patients, including T1-weighted, T2-weighted, and T1-weighted with injection of Gadolinium (T1W + Gd) images. Contrast stretching and median filter were applied to enhance image intensity and remove noise, and the pre-processed images were reconstructed into NIfTI format files for deep learning. The MRI images were then transformed to fit the CNN’s requirements. A 3D U-Net architecture was proposed with optimized parameters to build an automatic segmentation model capable of segmenting osteosarcoma from the MRI images. The 3D U-Net segmentation model achieved excellent results, with mean dice similarity coefficients (DSC) of 83.75%, 85.45%, and 87.62% for T1W, T2W, and T1W + Gd images, respectively. However, the study found that the proposed method had some limitations, including poorly defined borders, missing lesion portions, and other confounding factors. In summary, an automatic segmentation method based on a CNN has been developed to address the challenge of manually segmenting osteosarcoma cancerous cells in MRI images. While the proposed method showed promise, the study revealed limitations that need to be addressed to improve its efficacy.