Previous studies have reported beta-lactamase-mediated penicillin resistance in Fusobacterium nucleatum, but no beta-lactamase gene has yet been identified in this species. An F. nucleatum subsp. polymorphum strain resistant to penicillin and amoxicillin was isolated from a human periodontitis sample. DNA cloning and sequencing revealed a 765-bp open reading frame encoding a new class D beta-lactamase named FUS-1 (OXA-85). A recombinant Escherichia coli strain carrying the bla(FUS-1) gene exhibited resistance to amoxicillin with a moderate decrease in the MICs with clavulanic acid. The bla(FUS-1) gene was found in two additional clonally unrelated F. nucleatum subsp. polymorphum isolates. It was located on the chromosome in a peculiar genetic environment where a gene encoding a putative transposase-like protein is found, suggesting a possible acquisition of this class D beta-lactamase gene. The FUS-1 enzyme showed the closest ancestral relationship with OXA-63 from Brachyspira pilosicoli (53% identity) and with putative chromosomal beta-lactamases of Campylobacter spp. (40 to 42% identity). FUS-1 presents all of the conserved structural motifs of class D beta-lactamases. Kinetic analysis revealed that FUS-1 exhibits a narrow substrate profile, efficiently hydrolyzing benzylpenicillin and oxacillin. FUS-1 was poorly inactivated by clavulanate and NaCl. FUS-1 is the first example of a class D beta-lactamase produced by a gram-negative, anaerobic, rod-shaped bacterium to be characterized.