New insights into tumor-associated metabolic reprogramming have provided novel vulnerabilities that can be targeted for cancer therapy. Here, we propose a mass spectrometry imaging (MSI)-based metabolomic strategy to visualize the spatially resolved reprogramming of carnitine metabolism in heterogeneous breast cancer. Methods: A wide carnitine coverage MSI method was developed to investigate the spatial alternations of carnitines in cancer tissues of xenograft mouse models and human samples. Spatial expression of key metabolic enzymes that are closely associated with the altered carnitines was examined in adjacent cancer tissue sections. Results: A total of 17 carnitines, including L-carnitine, 6 short-chain acylcarnitines, 3 middle-chain acylcarnitines, and 7 long-chain acylcarnitines were imaged. L-carnitine and short-chain acylcarnitines are significantly reprogrammed in breast cancer. A classification model based on the carnitine profiles of 170 cancer samples and 128 normal samples enables an accurate identification of breast cancer. CPT 1A, CPT 2, and CRAT, which are extensively involved in carnitine system-mediated fatty acid β-oxidation pathway were also found to be abnormally expressed in breast cancer. Remarkably, the expressions of CPT 2 and CRAT were found for the first time to be altered in breast cancer. Conclusion: These data not only expand our understanding of the complex tumor metabolic reprogramming, but also provide the first evidence that carnitine metabolism is reprogrammed at both the metabolite and enzyme levels in breast cancer.