FOXO1, a member of the forkhead family of transcription factors, plays a vital role in the osteogenic lineage commitment of mesenchymal stem cells, and affects multiple cellular functions of osteogenic cells. However, prior studies have focused on mesenchymal stem cells but not on differentiated osteoblasts. In addition, studies about the role of FOXO1 during osseointegration are lacking. In this present study, we constructed osteoblast conditional FOXO1 knock-out mice and lentivirus-mediated FoxO1 overexpression to investigate maxillary titanium implant osseointegration. After 4 wk post implant placement, micro-computed tomography, histomorphometric analyses, and RT-qPCR assays were performed. Results showed that compared with the control group, overexpression of FOXO1 significantly enhanced bone formation around implant and bone-implant contact ratio, while loss of FOXO1 impaired peri-implant osteogenesis and osseointegration. Moreover, overexpression of FoxO1 enhanced expression of osteogenesis-related genes, such as Runx2, Alp1, Col1a1, and Bglap. Whereas, knock-out of Foxo1 reduced the expression of osteogenesis-related genes. Taken together, our results suggested that FOXO1 in osteoblasts could enhance osteogenesis-related gene expression to improve osseointegration.