To distinguish the influences of fuel type and truck speed on chemical composition and sub-toxic effects of particulates (PM2.5) from engine emissions, biomarkers—interleukin-6 (IL-6), cytochrome P450 (CYP) 1A1, heme oxygenase (HO)-1, and NADPH-quinone oxidoreductase (NQO)-1—were studied in A549 human lung cells. Fuel type and truck speed preferentially affected the quantity and ion/polycyclic aromatic hydrocarbon (PAH) composition of PM2.5, respectively. Under idling operation, phenanthrene was the most abundant PAH. At high speed, more than 50% of the PAHs had high molecular weight (HMW), of which benzo[a]pyrene (B[a]P), benzo[ghi]perylene (B[ghi]P), and indeno[1,2,3-cd]pyrene (I[cd]P) were the main PAHs. B[a]P, B[ghi]P, and I[cd]P caused potent induction of IL-6, CYP1A1, and NQO-1, whereas phenanthrene mildly induced CYP1A1. Based on the PAH-mediated induction, the predicted increases in biomarkers were positively correlated with the measured increases. HMW-PAHs contribute to the biomarker induction by PM2.5, at high speed, which was reduced by co-exposure to epigallocatechin-3-gallate.