Outside of their involvement in energy production, mitochondria play a critical role for the cell through their access to a discrete pathway for fatty acid biosynthesis. Despite decades of study in bacterial fatty acid synthases (the putative evolutionary mitochondrial precursor), our understanding of human mitochondrial fatty acid biosynthesis remains incomplete. In particular, the role of the key carrier protein, human mitochondrial acyl carrier protein (mACP), which shuttles the substrate intermediates through the pathway, has not been well-studied in part due to challenges in protein expression and purification. Herein, we report a reliable method for recombinant Escherichia coli expression and purification of mACP. Fundamental characteristics, including substrate sequestration and chain-flipping activity, are demonstrated in mACP using solvatochromic response. This study provides an efficient approach toward understanding the fundamental protein-protein interactions of mACP and its partner proteins, ultimately leading to a molecular understanding of human mitochondrial diseases such as mitochondrial fatty acid oxidation deficiencies.