C3larvinA is a putative virulence factor produced by Paenibacillus larvae enterobacterial-repetitive-intergenic-consensus (ERIC) III/IV (strain 11-8051). Biochemical, functional and structural analyses of C3larvinA revealed that it belongs to the C3-like mono-ADP-ribosylating toxin subgroup. Mammalian RhoA was the target substrate for its transferase activity suggesting that it may be the biological target of C3larvinA. The kinetic parameters of the NAD+ substrate for the transferase (KM = 75 ± 10 μM) and glycohydrolase (GH) (KM = 107 ± 20 μM) reactions were typical for a C3-like bacterial toxin, including the Plx2A virulence factor from Paenibacillus larvae ERIC I. Upon cytoplasmic expression in yeast, C3larvinA caused a growth-defective phenotype indicating that it is an active C3-like toxin and is cytotoxic to eukaryotic cells. The catalytic variant of the Q187-X-E189 motif in C3larvinA showed no cytotoxicity toward yeast confirming that the cytotoxicity of this factor depends on its enzymatic activity. A homology consensus model of C3larvinA with NAD+ substrate was built on the structure of Plx2A, provided additional confirmation that C3larvinA is a member of the C3-like mono-ADP-ribosylating toxin subgroup. A homology model of C3larvinA with NADH and RhoA was built on the structure of the C3cer-NADH-RhoA complex which provided further evidence that C3larvinA is a C3-like toxin that shares an identical catalytic mechanism with C3cer from Bacillus cereus. C3larvinA induced actin cytoskeleton reorganization in murine macrophages, whereas in insect cells, vacuolization and bi-nucleated cells were observed. These cellular effects are consistent with C3larvinA disrupting RhoA function by covalent modification that is shared among C3-like bacterial toxins.,? 2020 The Author(s).