A superoxide dismutase (SOD) was characterized from Beauveria bassiana, a fungal entomopathogen widely applied to insect control. This 209-aa enzyme (BbSod2) showed no more than 71% sequence identity to other fungal Mn-SODs, sharing all conserved residues with the Mn-SOD family and lacking a mitochondrial signal. The SOD activity of purified BbSod2 was significantly elevated by Mn2+, suppressed by Cu2+ and Zn2+ but inhibited by Fe3+. Overexpressing the enzyme in a BbSod2-absent B. bassiana strain enhanced its SOD activity (107.2 +/- A 6.1 U mg(-1) protein) by 4-10-fold in different transformants analyzed. The best BbSod2-transformed strain with the SOD activity of 1,157.9 +/- A 74.7 U mg(-1) was 93% and 61% more tolerant to superoxide-generating menadione in both colony growth (EC50 = 2.41 +/- A 0.03 versus 1.25 +/- A 0.01 mM) and conidial germination (EC50 = 0.89 +/- A 0.06 versus 0.55 +/- A 0.07 mM), and 23% more tolerant to UV-B irradiation (LD50 = 0.49 +/- A 0.02 versus 0.39 +/- A 0.01 J cm(-2)). Its virulence to Spodoptera litura larvae was enhanced by 26% [LT50 = 4.5 (4.2-4.8) versus 5.7 (5.2-6.4) days]. Our study highlights for the first time that the Mn2+-cofactored, cytosolic BbSod2 contributes significantly to the virulence and stress tolerance of B. bassiana and reveals possible means to improving field persistence and efficacy of a fungal formulation by manipulating the antioxidant enzymes of a candidate strain.