Malate Dehydrogenase (MDH), as a critical enzyme in cancer metabolism, can be the main foundation for NAD, along with the well-known LDH, which allows the prolonged process of glycolysis, even in the absence of oxygen. The kinetic characteristics of the enzyme are related to the surrounding environment of the enzyme. In this regard, the tumor microenvironment has noticeable properties, in connection with the neighboring normal tissues. The goal of this research is to elaborate the kinetic parameters of MDH in human breast cancer by taking into account two major perspectives as follows: 1. the possible role of MDH in supporting the NAD pool; 2. the impact of different tumor microenvironments on the kinetic characteristics of the enzyme. For this purpose, the MDH activity was measured from the normal and malignant crude human breast tissues, which were directly collected from the operating room. The Michaelis-Menten constant (Km) and the maximum velocity (Vmax) were determined in the crude extracts. It was realized that the affinity of cancerous MDH in the forward reaction was identical to the normal MDH, while the Vmax of cancerous MDH was higher than the normal MDH. In the reverse reaction, the affinity of cancerous MDH for malate and NAD+ was less than the normal MDH. The increasing affinity of MDH for malate and the decreasing MDH activity and expression in the forward reaction may be an acceptable molecular target for eliminating its possible impact on tumorigenesis. Therefore, the kinetic characteristics of MDH can be demonstrated as a novel diagnostic parameter for human breast cancers. However, further research seems necessary to validate this proposition. In addition, various kinetic parameters of different cancer cell lines should be investigated in the culture conditions, where both oxygen and nutrients are limited, similar to the tumor microenvironment.