This study sought to determine the role of receptor activator of nuclear factor-kappaB ligand (RANKL) in the Porphyromonas gingivalis infection-associated periodontal bone loss. Rowett rats were infected orally on Day 0–3 with 108 live Pg (ATCC 33277). Separate groups received anti-RANKL antibody, osteoprotegerin (OPG)-Fc, or control L6-Fc injection into gingival papilla in addition to P. gingivalis infection. After 4 weeks, robust immune responses to P. gingivalis were detected in infected rats. The majority of RANKL-expressing cells in rat gingival tissues are T and B cells. Soluble RANKL in gingival homogenates and periodontal bone loss were significantly elevated in P. gingivalis infected rats compared to uninfected rats (p < 0.05). Injection of anti-RANKL antibody or OPG-Fc into rat gingival papillae after P. gingivalis infection significantly reduced periodontal bone loss (p < 0.05). This study suggests that P. gingivalis infection-associated bone loss is mediated by immune cells in a RANKL-dependent manner.