Epidemiological studies have shown that exposure to Polycyclic aromatic hydrocarbons (PAHs) is associated with reduced mitochondrial DNA copy number (mtDNA-CN). Long non-coding RNA maternally expressed gene 3 (MEG3) is involved in mitochondrial function regulation. However, it is unknown whether single-nucleotide polymorphisms in the MEG3 can regulate the mtDNAcn in PAHs exposed populations. The aim of this study was to examine the effect of MEG3 genetic polymorphisms on the mtDNA-CN in PAHs exposed populations. We recruited 544 coke oven workers and 238 controls using random cluster sampling. High-performance liquid chromatography was used to detect the concentrations of four OH-PAHs (1-hydroxypyrene 1-OHPyr, 1-hydroxynathalene 1-OHNap, 2-hydroxynathalene 2-OHNap, and 3-hydroxyphenanthrene 3-OHPhe) in urine. The mtDNA-CN of peripheral blood leukocytes was measured using the quantitative polymerase chain reaction method. Sequenom Mass ARRAY matrix-assisted laser desorption/ionization-time of flight mass spectrometry platform was used to detect ten polymorphisms in MEG3. The OH-PAHs levels in the exposure group were significantly higher than those in the control group (P < 0.001). The mtDNA-CN in the exposure group was significantly lower than that in the control group (P < 0.001). A linear regression model revealed that PAHs-exposure (β 95% confidence interval, CI, −0.428 -0.475, −0.381, P < 0.001), male gender (−0.052 -0.098, −0.005, P = 0.029), genotype TT for MEG3 rs11859 (−0.088 -0.142, −0.035, P = 0.001), and genotype GG for MEG3 rs7155428 (−0.114 -0.210, −0.017, P = 0.021) were associated with decreased mtDNA-CN. PAHs-exposure, male gender, genotype TT for rs11859, and genotype GG for rs7155428 were risk factors for mtDNA-CN. Display omitted •Flight mass spectrometry platform was established to detect MEG3 polymorphisms.•PAHs exposure induced the mtDNA-CN decrease.•MEG3 rs11859 TT and rs7155428 GG may be risk factors of the reduction of mtDNA-CN.