There has been a recent emergence of epidemic spinal infections with necrosis causing lameness and mortality in male broilers and broiler breeders. Mortality in affected flocks may be as high as 15%. The disease has been called enterococcal spondylitis (ES), based on the frequent isolation of Enterococcus cecorum from the lesions and necrosis and inflammation observed in the free thoracic vertebrae (FTV) of affected birds. Male broiler breeders in an experimental setting were challenged with pure E. cecorum isolates obtained from ES-affected commercial flocks. Challenge routes included oral gavage (10⁸), intravenous (IV; 10³), and air sac (AS; 10³). Half the study birds in each group were chemically immunosuppressed with dexamethasone. Spinal lesions were observed grossly in birds challenged intravenously (2.9%) and birds challenged orally (6.1%). Microscopic spinal lesions consistent with ES were more frequently identified compared with gross lesions in the orally challenged group (30.3%). Chemical immunosuppression with dexamethasone was not associated with a greater incidence of ES in this study. By recreating the disease experimentally, the study design reported here may help in the further development of an experimental challenge model for future studies on risk factors, prevention, and therapeutic intervention of ES.