Abstract Because DIF-1 has been shown to affect Wnt/β-catenin signaling pathway, the effects of DIF-1 on osteoblast-like cell lines, SaOS-2 and MC3T3-E1, were examined. We found that DIF-1 inhibited this pathway, resulting in the suppression of ALP promoter activity through the TCF/LEF binding site. Introduction: Differentiation-inducing factor-1 (DIF-1), a morphogen of Dictyostelium, inhibits cell proliferation and induces cell differentiation in several mammalian cells. Previous studies showed that DIF-1 activated glycogen synthase kinase-3β, suggesting that this chemical could affect the Wnt/β-catenin signaling pathway. This pathway has been shown to be involved in bone biology. Materials and Methods: We studied the effects of DIF-1 on SaOS-2 and MC3T3-E1, osteosarcoma cell lines widely used as a model system for ostoblastic cells and murine osteoblast-like cell line, respectively. Reporter gene assays were also carried out to examine the effect of DIF-1 on the Wnt/β-catenin signaling pathway. Results: DIF-1 inhibited SaOS-2 proliferation and reduced alkaline phosphatase (ALP) activity in a concentration- and a time-dependent manner. The expression of ALP was markedly suppressed by DIF-1-treatment in protein and mRNA levels. DIF-1 also suppressed the expression of other osteoblast differentiation markers, including core binding factor α1, type I collagen, and osteocalcin, in protein and mRNA levels and inhibited osteoblast-mediated mineralization. Subsequently, we examined the effect of DIF-1 on the Wnt/β-catenin signaling pathway. We found that DIF-1 suppressed the expression of β-catenin protein and the activity of the reporter gene containing T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) consensus binding sites. We examined the effect of DIF-1 on a reporter gene driven by the human ALP promoter and found that DIF-1 significantly reduced the ALP reporter gene activity through the TCF/LEF binding site (−1023/−1017 bp). Furthermore, the effect of DIF-1 on MC3T3-E1, a murine osteoblast-like cell line, was examined, and it was found that DIF-1 suppressed ALP mRNA expression by the reduction of the ALP reporter gene activity through the TCF/LEF binding site. Conclusions: Our data suggest that DIF-1 inhibits Wnt/β-catenin signaling, resulting in the suppression of ALP promoter activity. To our knowledge, this is the first report to analyze the role of the TCF/LEF binding site (−1023/−1017 bp) of the ALP gene promoter in osteoblast-like cell lines.