Bovine tuberculosis results from infection with Mycobacterium bovis (M. bovis), a member of the M. tuberculosis complex. Worldwide, M. bovis infections result in economic losses in the livestock industry; cattle production is especially hard-hit by this disease. Generating M. bovis-resistant cattle could therefore potentially mitigate the impact of this disease by reducing M. bovis infections. In this study, we used transgenic somatic cell nuclear transfer (SCNT) to generate cattle expressing human β-defensin-3 (HBD3), which confers resistance to mycobacteria in vitro. We first generated alveolar epithelial cells (AECs) expressing HBD3 under the control of the bovine MUC1 promoter, and confirmed that these cells secreted HBD3 and possessed anti-mycobacterial capacity. We then generated and identified transgenic cattle by SCNT. The cleavage and blastocyst formation rates of genetically modified embryos provided evidence that monoclonal transgenic bovine fetal fibroblast cells have an integral reprogramming ability that is similar to that of normal cells. Five genetically modified cows were generated and identified, and their anti-mycobacterial capacities were evaluated. AECs and macrophages from these cattle expressed higher levels of HBD3 protein compared with normal cells and possessed effective anti-mycobacterial capacity. These results suggest that the overall risk of M. bovis infection in transgenic cattle is efficiently reduced and support the development of genetically modified animals as an effective tool to reduce M. bovis infection. This article is protected by copyright. All rights reserved.