Background: Osteosarcoma, the most common primary tomour of bone, is the second highest cause of cancer-related deaths in the paediatric age group. Cure rates for patients with metastatic or recurrent disease remain poor (<20% survival). Constitutive STAT3 activation is associated with various human cancers and commonly suggests poor prognosis [1]. Thus STAT3 has been studied as a tumour therapeutic target. Our previous data showed that constitutive activation of STAT3 was detected in a broad range of human soft tissue sarcoma (STS) cell lines. In addition, we demonstrated that STAT3 inhibitor S3I-201 induced anti-proliferative effects on majority of STS cell lines harbouring aberrant STAT3 by blocking STAT3 phosphorylation [2]. This study aimed to investigate the effect of STAT3 inhibitor S3I-201 therapy on growth of a panel of 6 osteosarcoma cell lines. Methods: STAT3 inhibitor S3I-201 mono-therapy was investigated in a panel of 6 osteosarcoma cell lines (Saos-2, U2-OS, HOS, SJSA, 143B and MG63). The effect of S3I-201 was assessed by both crystal-violet colorimetric and clonogenic assays. Results: Anti-proliferative effects of S3I-201 mono-therapy were dose- and time-dependent. STAT3 inhibitor alone had anti-tumour growth effect with different mean IC50s (143B: 51µM, HOS: 40 µM, MG63: 84 µM, SJSA: 72 µM, U2-OS: 30 µM and Saos-2: 27 µM). Colony formation ability following drug treatment was also investigated by clonogenic assay. Consistently, at treatment with 25-50 µM S3I-201, majority of osteosarcoma cell lines had completely abrogated colony formation ability. Conclusion: Our study demonstrated that targeting STAT3 using S3I-201 suppressed human osteosarcoma cell growth in vitro. Further studies will focus on potential mechanisms as well as the anti-sarcoma effect in the animal models.