MicroRNAs regulate target gene expression and are involved in cell proliferation, apoptosis, differentiation, tumor invasion, and cancer stem cell regulation, among other processes. MicroRNA-26b (miR-26b) is closely related to tumor occurrence and development. In this study, we analyzed miR-26b expression in osteosarcoma tissue, its effect on Saos-2 osteosarcoma cell proliferation and invasion, and its relationship with 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) expression. Osteosarcoma tissue was obtained from surgical patients and normal tissue adjacent to the tumor was used as a control. Real-time polymerase chain reaction was applied to detect miR-26b expression in cancer tissue and normal tissue. A vector expressing miR-26b was constructed and transfected into Saos-2. An MTT assay, cell invasion assay, and scratch experiment were used to analyze the effect of miR-26b on Saos-2 cell proliferation, invasion, and migration abilities. Western blotting analysis was performed to investigate the role of miR-26b on PFKFB3 expression. miR-26b expression in normal tissue was 7.55-fold higher than in osteosarcoma tissue (t = 10.20, P = 0.006). Compared with control tissue, miR-26b significantly inhibited osteosarcoma proliferation, migration, and invasion (P < 0.05). Western blotting results revealed that PFKFB3 protein expression decreased in Saos-2 cells after transfection with miR-26b. miR-26b was down-regulated in osteosarcoma tissue. miR- 26b may inhibit osteosarcoma cell proliferation, migration, and invasion by regulating PFKFB3 protein expression. miR-26b may have a tumor suppressor role in tumor occurrence and development.