Objective To study the relationship between polymorphism of NAT2 gene and the susceptibility of osteosarcoma. Methods Using serum samples of patients with osteosarcoma , NAT2 gene polymorphism was determined by PCR-RFLP to observe M1, M2 and M3 mutant genes, NAT2 genotype and allele distribution. Slow acetylation and rapid acetylation genotype between groups in the distribution were also detected. Moreover , the relationship between the clinical characteristics of osteosarcoma and NAT2 genetic polymorphism were analyzed. Results From January 2010 to September 2015, 126 patients with osteosarcoma were enrolled as a case group, and 119 healthy persons in the same period were as the control group. In the control group, the frequency of NAT2 genotype (homozygous wild type (wt/wt and miscellaneous synthetic mutant WT/ MX, homozygous mutant MX/MX) were 30.95%, 50.79% and 18.25% respectively; In the experimental group were 47.06%, 46.22% and 6.72% respectively. The frequency of the two groups was statistically significant (P <0.05). Rapid NAT2 acetylator genotype of suffering from the risk of osteosarcoma 1.782 times slow acetylation genotype (P < 0.05). Compared to rapid acetylation genotype patients with osteosarcoma, slow acetylation genotype patients has smaller tumor volume (P = 0.008),lower differentiation degree of tumor (P = 0.011) and less occurrence of distant metastasis (P = 0.001). Conclusion The rapid acetylation genotype of NAT2 gene may be a risk factor for osteosarcoma.