The entomopathogenic fungus Metarhizium anisopliae MA05-169 is a transformant strain that has been metabolically engineered to express dihydroxynaphthalene-melanin biosynthesis genes. In contrast to the wild type strain, the transformant displays a greater resistance to environmental stress and a higher virulence toward target insect host. However, the underlying mechanisms for these characteristics remain unclear; hence experiments were initiated to explore the possible mechanism(s) through physiological and molecular approaches. Although both transformant and wild type strains could infect and share the same insect host range, the former germinated faster and produced more appressoria than the latter, both in vivo and in vitro. The transformant showed a significantly shorter median lethal time (LT50) when infecting the diamondback moth (Plutella xylostella) and the striped flea beetle (Phyllotreta striolata), than the wild type. Additionally, the transformant was more tolerant to reactive oxygen species (ROS), produced 40-fold more orthosporin and notably overexpressed the transcripts of the pathogenicity-relevant hydrolytic enzymes (chitinase, protease, and phospholipase) genes in vivo. In contrast, appressorium turgor pressure and destruxin A content were slightly decreased compared to the wild type. The transformant's high anti-stress tolerance, its high virulence against five important insect pests (cowpea aphid Aphis craccivora, diamondback moth Pl. xylostella, striped flea beetle Ph. striolata, and silverleaf whitefly Bemisia argentifolii) and its capacity to colonize the root system are key properties for its potential bio-control field application.