In crustaceans, anti-lipopolysaccharide factors (ALFs) are important immune effectors that have sequence diversity and exhibit broad antimicrobial activities. In this study, we characterized a novel ALF homolog SpALF6 from mud crab Scylla paramamosain and its variant SpALF6-V, which was generated by mutations of two amino acids (H46 to R and A110 to P) due to the presence of two single nucleotide polymorphisms (SNPs). SpALF6 was an anionic peptide with isoelectric point (pI) 6.79, whereas SpALF6-V was a cationic protein with pI 7.98. These two proteins shared a common lipopolysaccharide (LPS)-binding domain (LBD) with pI 6.05. SpALF6 was expressed mainly in hemocytes and up-regulated by Vibrio parahaemolyticus or Staphylococcus aureus challenge, indicating that SpALF6 may participate in the antibacterial immune responses. To investigate the likely functional differences between SpALF6 and SpALF6-V and elucidate the underlying mechanisms, a single amino acid mutant SpALF6-M (from H46 to R, outside but very close to LBD), which had the same pI as SpALF6-V, was harvested by a fusion PCR. Then, both SpALF6 and SpALF6-M were overexpressed and purified to test antimicrobial activity and binding activity to microbial cells or polysaccharides. SpALF6-M exhibited more potent antimicrobial and cell-binding activity on Gram-positive bacteria and fungi than SpALF6. Furthermore, SpALF6-M possessed stronger lipoteichoic acid (LTA)-binding activity than SpALF6, demonstrating that this particular positively charged amino acid outside but close to LBD contributed to the increase in SpALF6-M antibacterial activity. In addition, SpALF6 LBD peptide and its biotin-labeled form were synthesized in this study. Results showed that this anionic LBD peptide itself did not exhibit any significant antimicrobial activity against 10 kinds of microorganisms but it possessed strong binding activity to LPS, LTA, and peptidoglycan. These findings suggested that this anionic LBD was still an important active center and required collaboration with some particular positively charged amino acids outside LBD to exhibit antibacterial activity. Thus, SpALF6-M antimicrobial activity was increased by the mutation of H46 to R instead of A110 to P, which did not change the protein charge, suggesting that SpALF6-V may have more potent antimicrobial activity than SpALF6 and play more important roles in antibacterial immunity. This study provided a new insight into the mechanisms of how ALF amino acid sequence diversity resulted in their functional divergence.