Escherichia coli YajC is a small integral membrane protein with a single transmembrane helix. The gene yajC is part of the secD operon and the protein is identified in the SecDF-YajC complex. However, the exact function of YajC remains a mystery. While its function is usually discussed in the context of the SecDF-YajC complex, studies have shown that SecD/F, rather than YajC, are essential for those functions. Recently YajC is identified as the mysterious protein that co-crystallized with AcrB. To further investigate the structure of YajC, we expressed and purified the protein in a detergent solubilized state. The protein assumed a folded structure containing mixed α/β secondary structures, consistent with the structural prediction. Using signal Cys mutations and thiol-specific probes, we found the C-terminus of YajC was cytoplasmic, while the N-terminus of YajC was buried in the membrane. In addition, we expressed and purified a truncated fragment of YajC that corresponded to the C-terminal cytoplasmic domain (YajC(CT)). YajC(CT) formed a compact structure rich in β-strands and existed as a trimer.