Splenectomy is sometimes required in bone fracture patients. The present study aims to investigate the effect of splenectomy on fracture healing in rats.Rats underwent osteotomy were subjected to splenectomy. The effects of splenectomy were evaluated at day 0, 3, 15 and 30 post-fracture. Double immunofluorescence staining was used to examine the expression of macrophages marker F4/80 and CD11b. H&E staining was used to examine the histological changes in fracture sites. Western blotting was used to detect protein expression of osteoprotegerin (OPG), the ligand for receptor activator of NF-κB (RANKL) and collagens in the fracture site. Activity of alkaline phosphatase (ALP) in the serum was determined using a biochemical kit. Serum levels of osteocalcin and inflammatory cytokines were determined using ELISA kits. Real-time PCR was used to detect mRNA expression of ALP and osteocalcin in the fracture site.Results showed that the recruitment of macrophages and the production of inflammatory cytokines were inhibited in the fracture rats underwent splenectomy. Importantly, histological examination showed that fracture healing was delayed in splenectomized rats. In addition, the protein expression of OPG and RANKL in the fracture site was diminished, the activity of ALP and the level of osteocalcin in the serum and their mRNA expression in the fracture site were reduced, and the protein expression of type I collage a1 and type II collage a1 was inhibited in fracture rats underwent splenectomy compared with that in rats without splenectomy.Our findings indicate that splenectomy delays fracture healing.