Abstract: Anti-lipopolysaccharide factors (ALFs), a type of the potent antimicrobial peptide, can bind and neutralize lipopolysaccharide (LPS) and exhibit broad spectrum antimicrobial activities. In order to study the function of ALFs in congenital immunization of Macrobrachium nipponense, two isoforms of the ALF homologues (MnALF1 and MnALF2) were cloned and characterized from the oriental river prawn M. nipponense. The full-length cDNA sequences of MnALF1 and MnALF2 were 1008 and 836 bp, encoding 121 and 124 amino acids, respectively. All of these sequences contained one signal peptide and an LPS-binding domain with two conserved cysteine residues at both ends of the domain. The deduced peptide of MnALF1 and MnALF2 was highly similar to previously identified ALFs in crustaceans. qRT-PCR showed that MnALFs were expressed in all detected tissues. MnALF1 transcript was predominantly detected in heart and intestine and MnALF2 transcript was predominantly detected in hemocytes and hepatopancreas. After challenge with Aeromonas hydrophila, two MnALF transcripts in heart, intestine, hemocytes and hepatopancreas showed a clear time-dependent response expression pattern (the expression levels of MnALF1 present an trend of down-regulated first and then increasing, which reached to the highest level at 24h, 12h, 36h, 24h in heart, intestine, hemocytes and hepatopancreas respectively. However, for MnALF2, the expression level present an down-regulated trend and then increased in heart, intestine; In hemocytes and hepatopancreas, the expression level present an increasing trend and then down-regulated. MnALF2 transcripts reached to the top at 24h post-challenge). These results suggest that two MnALF isoforms have different tissue specificity and might provide multiple protective functions in immune defense against invading bacteria.