Apolipoprotein A-I is an anti-inflammatory, antioxidative, cardioprotective, anti-tumorigenic, and anti-diabetic in mammals. Apolipoprotein A-I also regulates innate immune defense mechanisms in vertebrates and invertebrates. Apolipoproteins A-I from mammals and several teleosts display antibacterial activities against Gram negative and Gram positive bacteria. The present study describes strategies to obtain high amounts of soluble purified recombinant Apolipoprotein A-I of Labeo rohita, an Indian major carp (rLrApoA-I). The study also reports its detailed structural and functional characterization i.e. antimicrobial activity against a number of important marine and fresh water bacterial pathogens. The rLrApoA-I was expressed in Escherichia coli BL21(DE3) pLysS expression host as a soluble protein under optimized conditions. The yield of purified rLrApoA-I was ~ 75 mg/L from soluble fraction using metal ion affinity chromatography. The authenticity of the rLrApoA-I was confirmed by MALDI-TOF-MS analysis. The secondary structure analysis showed rLrApoA-I to be predominantly alpha helical, an evolutionary conserved characteristic across mammals and teleosts. The purified rLrApoA-I exhibited antimicrobial activity as evident from inhibition of growth of a number of bacteria namely Aeromonas hydrophila, A. liquefaciens, A. culicicola, A. sobria, Vibrio harveyi, V. parahaemolyticus and Edwardsiella tarda in a dose-dependent manner. Minimum bactericidal concentration for A. liquefaciens, A. culicicola, and A. sobria, was determined to be 25 μg/ml or 0.81 μM whereas for A. hydrophila, E. tarda, V. parahaemolyticus and V. harveyi, it was determined to be 100 μg/ml or 3.23 μM. These data strongly suggest that recombinant ApoA-I from Labeo rohita could play a role in primary defense against fish pathogen. Further, at temperature ≥ 55 °C, though a loss in secondary structure was observed, no effect on its antibacterial activity was observed. This is of significance as the antibacterial activity is not likely to be lost even if the protein is subjected to high temperatures during transport.