Osteosarcoma is the most common type of bone cancer. In the present study, by way of PCR-based microarrays, we found that TUT1, a nucleotidyl transferase, was significantly downregulated in osteosarcoma, compared with adjacent normal tissues. In the current study, we performed PCR-based microarrays using the cDNA prepared from osteosarcoma and adjacent normal tissues. The enforced expression of TUT1 was able to inhibit cell proliferation in U2OS and MG63 cells, while its knockdown using small interfering RNA (siRNA) oligos promoted cell proliferation. At the molecular level, we found that TUT1 could inhibit the expression levels of PPARgamma and SREBP-1c, two key regulators in lipogenesis, through upregulation of microRNA-24 and microRNA-29a. Therefore, our results suggest that TUT1 may act as a tumor suppressor for osteosarcoma, which might provide a novel mechanism for the tumor development.