The present study aimed to investigate the effect of β-receptor blocker propranolol on early osseointegration of pure titanium implants and the underlying molecular regulatory mechanisms. An implant osseointegration model using the tibial metaphysis of New Zealand rabbits was established. The rabbits were divided into control and low-, medium-and high-dose propranolol groups. The formation of implant osseointegration was detected by X-ray scanning. Mesenchymal stem cells (MScs) and osteoblasts (OBs) were isolated and cultured in vitro, isoproterenol was supplemented to simulate sympathetic action and propranolol was subsequently administrated. The effect of propranolol on cell proliferation and osteogenic differentiation were assessed by EdU, flow cytometry, alizarin red staining and alkaline phosphatase (ALP) detection. The expression levels of bone morphogenetic protein (BMP)2, RUNX family transcription factor (RunX)2, collagen (cOL)-1, osteocalcin (OcN) and β2-adrenergic receptor (AR) were detected by immunofluorescence, reverse transcription-quantitative PcR and western blot assay. Propranolol effectively promoted implant osseointegration in vivo, facilitated proliferation of OBs, inhibited proliferation of MScs and enhanced osteogenic differentiation of OBs and MScs. The calcium content and ALP activity of cells treated with propranolol were markedly higher than in the control group. Propranolol also elevated mRNA and protein expression levels of BMP2, RunX2, cOL-1 and OcN in tissue and cells, and decreased the expression of β2-AR. The present study demonstrated that the β-receptor blocker propranolol promoted osteogenic differentiation of OBs and MScs and enhanced implant osseointegration. The present study provided a novel insight into the application and regulatory mechanisms of propranolol.