This study focuses on the components and levels of polycyclic aromatic hydrocarbons (PAHs) and their derivatives (MPAHs and OPAHs) in plasma samples from 19 oil workers, pre- and post-workshift, and their exposure–response relationship with mitochondrial DNA (mtDNA) methylation. PAH, MPAH, OPAH, and platelet mtDNA methylation levels were determined using a gas chromatograph mass spectrometer (GC-MS) and a pyrosequencing protocol, respectively. The total plasma concentrations of PAHs in mean value were, respectively, 31.4 ng/mL and 48.6 ng/mL in pre- and post-workshift, and Phe was the most abundant (13.3 ng/mL in pre-workshift and 22.1 ng/mL in post-workshift, mean value). The mean values of total concentrations of MPAHs and OPAHs in the pre-workshift were 2.7 ng/mL and 7.2 ng/mL, while in the post-workshift, they were 4.5 ng/mL and 8.7 ng/mL, respectively. The differences in the meanMT-COX1,MT-COX2, andMT-COX3methylation levels between pre- and post-workshift were 2.36%, 5.34%, and 0.56%. Significant (p< 0.05) exposure–response relationships were found between PAHs and mtDNA methylation in the plasma of workers; exposure to Anthracene (Ant) could induce the up-regulation of the methylation ofMT-COX1(β= 0.831, SD = 0.105,p< 0.05), and exposure to Fluorene (Flo) and Phenanthrene (Phe) could induce the up-regulation of methylation ofMT-COX3(β= 0.115, SD = 0.042,p< 0.05 andβ= 0.036, SD = 0.015,p< 0.05, respectively). The results indicated that exposure to PAHs was an independent factor influencing mtDNA methylation.