The present study describes the discovery of Ph-Def, the first marine antimicrobial peptide (AMP) isolated from Panulirus homarus, the Indian spiny lobster. Ph-Def is a 65-amino acid β-defensin (including signal peptide) with potential antimicrobial properties. Phylogenetic analysis revealed its close relationship to β-defensins in other Panulirus lobsters and suggested an evolutionary connection with vertebrate β-defensins. In silico analysis identified hot spot areas and a hydrophobic face, supporting its potential as a marine AMP with antimicrobial activity. A predicted bactericidal stretch spanning from Cys53–Tyr64 was notably identified, covering the mature peptide. Ph-Def demonstrated similarity to β-defensins in other lobster species and fish β-defensins, suggesting a shared functional role against microorganisms. Predicted biological processes associated with Ph-Def include metabolic regulation, cellular biosynthesis, transport, cation transmembrane transport, and immune system processes. These findings underscore the potential of Ph-Def as a marine AMP and expand our understanding of immune responses and defense mechanisms in P. homarus.