The study objective was to investigate the effect of trichorhinophalangeal syndrome 1 (TRPS1) gene expression on multidrug-resistance of osteosarcoma and to reveal the mechanism. PCR and Western Blot were performed to determine the expression of TRPS1 and multidrug resistance 1 (MDR1) in U2-OS, MG-63, Saos-2 and U2-OS-siTRPS1 (U2-OS cells with inhibited expression of TRPS1) cell lines. Immunohistochemistry was used to determine the correlation between TRPS1 and MDR1 expressions in 63 cases of osteosarcoma tissues. Cholecystokinin- 8 (CCK-8) assay was performed to compare the chemotherapeutic drug-resistance of U2-OS and U2-OS-siTRPS1 cells. TRPS1 and MDR1 were most highly expressed in U2-OS cells, and less expressed in MG-63 cells. There were no P-gp proteins detected in Saos-2 cells. Correlation analysis showed that the expressions of TRPS1 and MDR1 in osteosarcoma cell lines were significantly correlated (P<0.001). Immunohistochemical staining of human osteosarcoma tissues showed that the expression of TRPS1 was positively correlated with the expression of P-gp in human osteosarcoma tissues (R-2=0.9658). siRNA mediated inhibition of TRPS1 in U2-OS cells resulted in significantly down-regulated expression of MDR1 (P<0.05) as well as significantly decreased the resistance to chemotherapeutic drugs such as adriamycin, methotrexate, paclitaxel and carboplatin (RI<0.5). The TRPS1 gene modulates drug-resistance of osteosarcoma cells by regulating the multidrug-resistance gene (MDR1) of osteosarcoma. Inhibition of TRPS1 expression could reduce the resistance to chemotherapeutic drugs of osteosarcoma cells.