The bone remodelling process is highly regulated by the immune response. Recent studies have shown that sphingosine-1-phosphate (S1P) is crucial in bone homeostasis, which functions through binding with its receptors (known as sphingosine-1-phosphate receptor 1 to 5, S1PR1-5) in the way of regulating immune cell migration and function, especially inducing the expression of nuclear factor factor-kappa B ligand (RANKL)—the key factor in osteoclastogenesis. Periapical lesions (due to tooth infection), which cause alveolar bone resorption and lesion formation around the tooth root tip, are an immune response to pathogen invasion resulted by osteoclast activation and imbalanced bone remodelling. Our study aims to investigate the expression of S1P and its receptor in periapical lesions and to unveil its possible role in the pathogenesis of periapical lesion. Our results showed that the expression of S1P and S1PR1 was enhanced in human periapical lesions. The up-regulated S1P-S1PR1 signalling was related with over-produced RANKL in the lesions, indicating this signaling might lead to osteoclastogenesis and bone loss; which was further identified in a rat periapical lesion model. Modulating S1P could down-regulate RANKL production and reduce osteoclastogenesis, thereby suppress the inflammatory bone destruction in periapical lesions. S1P is therefore a contributing factor in the pathogenesis of periapical lesions and could be a potential target for infection-induced bone loss management.