Endogenous molecules released by dying cells [i.e., damage-associated molecular patterns (DAMPs)] after trauma and severe blood loss can activate pattern recognition receptors, leading to a cascade of inflammatory responses and organ injury. Mitochondrial transcription factor A (TFAM) is a transcription factor for mitochondrial DNA. TFAM is structurally related to high mobility group box 1 (HMGB1), an important member of DAMPs. We, therefore, hypothesized that TFAM can be released into the circulation after hemorrhage to initiate inflammatory responses. In order to examine this hypothesis, male Sprague-Dawley rats were bled to and maintained at a mean arterial pressure of 40 mmHg for 90 min. They were then resuscitated with an equal volume of shed blood in the form of Ringer's lactate (i.e., low-volume resuscitation) over 60 min. TFAM levels in the serum were measured at 4 h after hemorrhage and resuscitation. Our results showed that serum levels of TFAM were more than doubled after hemorrhage and resuscitation. To further characterize TFAM's biological activity, we expressed recombinant rat TFAM with a GST-tag (GST-TFAM) in an E. coli expression system. The purity of GST-TFAM was over 99% and it was immunoreactive for specific anti-TFAM antibodies. Using RAW 264.7 cells and primary rat peritoneal macrophages, we showed that GST-TFAM dose-dependently increased TNF-α release. To determine the biological activity of GST-TFAM in vivo, GST-TFAM was intravenously injected in healthy male adult rats. Our results demonstrated that intravenous injection of GST-TFAM, not GST alone, upregulated circulating levels of pro-inflammatory cytokines, increased neutrophil infiltration to the lungs and caused organ injury in healthy animals. Thus, TFAM can act as a DAMP and may contribute to the initiation of inflammatory responses in hemorrhagic shock.