The intracellular fatty acid binding proteins (FABPs) are small soluble proteins (14-15 kDa) that can bind various fatty acids (≥ 14 carbons) with high affinity. These proteins have been found in both vertebrates and invertebrates. Recently, a novel FABP (lcFABP) which constituted about 15% of total soluble proteins in the light organ of adult firefly was found, implying a strong correlation between lcFABP and continuous bioluminescence flashes. Nevertheless, characteristics and functions of this lcFABP remain intriguing. Here, we overexpressed this protein in E. coli and investigated its structural and biochemical properties. Theb-strand conformation is dominated in lcFABP. In vitro binding assay showed that the lcFABP exhibited high affinity (average Kd <1 mM) to saturated fatty acids (C14-C18). In addition, the NMR results revealed that lcFABP was able to form a well-packed tertiary structure and certain residues in lcFABP were essential for the luciferin association. Accordingly, our studies suggest that lcFABP not only plays an important role in fatty acid transfering and utilization, but also participates in bioluminescence generating reaction through the luciferin regulation.