Neurogenic heterotopic ossifications (NHO) are heterotopic bones that develop in periarticular muscles after severe central nervous system (CNS) injuries. Several retrospective studies have shown that NHO prevalence is higher in patients who suffer concomitant infections. However, it is unclear whether these infections directly contribute to NHO development or reflect the immunodepression observed in patients with CNS injury. Using our mouse model of NHO induced by spinal cord injury (SCI) between vertebrae T₁₁ to T₁₃, we demonstrate that lipopolysaccharides (LPS) from gram-negative bacteria exacerbate NHO development in a toll-like receptor-4 (TLR4)-dependent manner, signaling through the TIR-domain-containing adapter-inducing interferon-β (TRIF/TICAM1) adaptor rather than the myeloid differentiation primary response-88 (MYD88) adaptor. We find that T₁₁ to T₁₃ SCI did not significantly alter intestinal integrity nor cause intestinal bacteria translocation or endotoxemia, suggesting that NHO development is not driven by endotoxins from the gut in this model of SCI-induced NHO. Relevant to the human pathology, LPS increased expression of osteoblast markers in cultures of human fibro-adipogenic progenitors isolated from muscles surrounding NHO biopsies. In a case–control retrospective study in patients with traumatic brain injuries, infections with gram-negative Pseudomonas species were significantly associated with NHO development. Together these data suggest a functional association between gram-negative bacterial infections and NHO development and highlights infection management as a key consideration to avoid NHO development in patients.