The mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1 is a ~97 kDa protein sharing sequence homology within the N-terminus with the catalytic domains of various bacterial ADP-ribosyltransferases. Here we studied the proteolytic activation of the ADP-ribosyltransferase activity of MTX. Chymotrypsin treatment of the 97 kDa MTX holotoxin (MTX30-870) results in a 70 kDa putative binding component (MTX265-870) and a 27 kDa enzyme component (MTX30-264), possessing ADP-ribosyltransferase activity. Chymotryptic cleavage of a N-terminal 32 kDa fragment of MTX (MTX30-308) also yields MTX30-264 but the resulting ADP-ribosyltransferase activity is much greater compared to the processed MTX30-870. Kinetic studies revealed a Km, NAD value of 45 µM for the processed 32 kDa MTX fragment, and a Km, NAD value for the processed holotoxin of 1300 µM. Moreover, the kcat value for the activated MTX30-308 fragment was about 10-fold higher than for the activated holotoxin (MTX30-870). Precipitation analysis showed that the 70 kDa proteolytic fragment of MTX remains non-covalently bound to the N-terminal 27 kDa fragment thereby inhibiting ADP-ribosyltransferase and NAD glycohydrolase activities. Glu197 of MTX30-264 was identified as the “catalytic” glutamate which is conserved in all ADP-ribosyltransferases. Whereas mutated MTX30-264E197Q has neither ADP-ribosyltransferase nor NAD glycohydrolase activities, mutated MTX30-264E195Q possesses glycohydrolase activity but not transferase activity. Transfection of HeLa cells with a vector encoding a fusion protein of MTX30-264 with a green fluorescent protein led to cytotoxic effects characterized by cell rounding and formation of filopodia-like protrusions. This cytotoxic effects were not observed with the catalytically inactive MTX30-264E197Q mutant, indicating that the MTX enzyme activity is essential for the cytotoxicity in mammalian cells.