Tuberculosis (TB) is a complex infectious disease caused by the pathogen Mycobacterium tuberculosis (Mtb) which has coexisted with humanity since the Neolithic. Recent research indicated that SIRT3 plays a pivotal role in promoting the antimycobacterial response of mitochondria and autophagy during Mtb infection. A case–control study comprised 900 TB patients and 1534 healthy controls who were retrospectively enrolled to assess the association between Sirt3 gene polymorphisms and TB susceptibility. In total, five single-nucleotide polymorphisms (SNPs) (rs511744, rs3782118, rs7104764, rs536715 and rs28365927) were selected through database 1000 Genomes Project and offline software Haploview V4.2 and genotyped by a customized 2 × 48-Plex SNPscan™ Kit. Our results suggested that the minor allele genotypes (A carriers) of rs3782118 confers the decreased risk of TB susceptibility (p_Bonferroni = 0.032), and a similar but more significant effect was observed under the dominant model analysis (OR 0.787, 95% CI 0.666–0.931, p_Bonferroni = 0.026). Haplotype analysis showed that haplotype AGAAG (rs511744/rs3782118/rs7104764/rs536715/rs28365927) was associated with an increased risk of TB (p = 0.023, OR 1.159, 95% CI 1.019–1.317). In stratification analysis, we found that rs3782118 was associated with decreased risk of TB in female subgroup under the dominant model analysis (p_Bonferroni = 0.016, OR 0.678, 95% CI 0.523–0.878). Moreover, functional annotations for three loci (rs7930823, rs3782116 and rs3782115) which are strongly linked to rs3782118 indicated that they may be responsible for the changes in some motifs. In conclusion, our study suggested that the SNP rs3782118 was associated with a lower susceptibility to TB, especially under the dominant model analysis and that the haplotype AGAAG (containing the major allele G of rs3782118) was associated with an increased risk of TB. Further independent cohort studies are necessary to validate the protective effect of Sirt3 genetic variants on the risk of TB.