Thiazolidinediones are traditional anti-diabetic therapeutic agents that have been associated with bone loss and increased fracture risk. However, the underlying mechanisms of this side effect require further elucidation. The present study aimed to investigate the effect of pioglitazone (PIO), a thiazolidinedione, on osteoblastogenesis, osteoclastogenesis and the osteoprotegerin (OPG) / receptor activator of nuclear factor-B ligand (RANKL) / RANK system. The MC3T3-E1 murine pre-osteoblastic cell line was treated with PIO and processed for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of OPG, RANKL, peroxisome proliferator-activated receptor (PPAR), Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN), and western blotting analysis of OPG and RANKL. The culture medium was collected for ELISA analysis of OPG and RANKL. Murine bone marrow monocytes (BMMCs) were treated with PIO in the presence of RANKL and macrophage-colony stimulating factor and subjected to tartrate-resistant acid phosphatase (TRAP) staining and activity measurement, and RT-qPCR analysis of cathepsin K, TRAP and RANK. Co-culture of MC3T3-E1 and BMMCs was performed in the presence of PIO, and TRAP staining was also conducted. PIO inhibited the osteoblastic differentiation of MC3T3-E1 cells, and promoted the osteoclastic differentiation of BMMCs with or without co-culturing with MC3T3-E1 cells. ELISA analysis indicated increased RANKL and decreased OPG expression levels in the medium of MC3T3-E1 cells treated with PIO. PIO upregulated expression of RANKL and PPAR and downregulated expression of OPG, RUNX2, ALP and OCN in MC3T3-E1 cells, while expression levels of RANK in BMMCs remained unchanged. These results suggest that PIO suppresses osteoblastogenesis and enhances osteoclastogenesis. In addition, PIO may also promote osteoclastogenesis by affecting the OPG-RANKL-RANK system.