Bovine tuberculosis (bTB) is a zoonotic disease in cattle that is transmissible to humans, distributed worldwide, and considered endemic throughout much of England and Wales. Mid-infrared (MIR) analysis of milk is used routinely to predict fat and protein concentration, and is also a robust predictor of several other economically important traits including individual fatty acids and body energy. This study predicted bTB status of UK dairy cows using their MIR spectral profiles collected as part of routine milk recording. Bovine tuberculosis data were collected as part of the national bTB testing program for Scotland, England, and Wales; these data provided information from over 40,500 bTB herd breakdowns. Corresponding individual cow life-history data were also available and provided information on births, movements, and deaths of all cows in the study. Data relating to single intradermal comparative cervical tuberculin (SICCT) skin-test results, culture, slaughter status, and presence of lesions were combined to create a binary bTB phenotype labeled 0 to represent nonresponders (i.e., healthy cows) and 1 to represent responders (i.e., bTB-affected cows). Contemporaneous individual milk MIR spectral data were collected as part of monthly routine milk recording and matched to bTB status of individual animals on the single intradermal comparative cervical tuberculin test date (±15 d). Deep learning, a sub-branch of machine learning, was used to train artificial neural networks and develop a prediction pipeline for subsequent use in national herds as part of routine milk recording. Spectra were first converted to 53 × 20-pixel PNG images, then used to train a deep convolutional neural network. Deep convolutional neural networks resulted in a bTB prediction accuracy (i.e., the number of correct predictions divided by the total number of predictions) of 71% after training for 278 epochs. This was accompanied by both a low validation loss (0.71) and moderate sensitivity and specificity (0.79 and 0.65, respectively). To balance data in each class, additional training data were synthesized using the synthetic minority over sampling technique. Accuracy was further increased to 95% (after 295 epochs), with corresponding validation loss minimized (0.26), when synthesized data were included during training of the network. Sensitivity and specificity also saw a 1.22- and 1.45-fold increase to 0.96 and 0.94, respectively, when synthesized data were included during training. We believe this study to be the first of its kind to predict bTB status from milk MIR spectral data. We also believe it to be the first study to use milk MIR spectral data to predict a disease phenotype, and posit that the automated prediction of bTB status at routine milk recording could provide farmers with a robust tool that enables them to make early management decisions on potential reactor cows, and thus help slow the spread of bTB., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)