Due to widely commercial applications of silver nanoparticles (Ag NPs), toxicity assessment of this NP is of great importance. This study aimed to investigate the oxidative stress and heat shock response of Ag NPs at different doses to A549 and HepG2 cells. After treatment with different concentrations of Ag NPs for 24 h, oxidative damage indicated by malondialdehyde (MDA), 8-epi-PGF2α, and 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) concentrations and protein levels of heat shock protein A1A (HSPA1A) and heme oxygenase 1 (HO-1) were determined. Ag NPs induced dose-dependent increases in MDA, 8-epi-PGF2α, and 8-oxo-dG concentrations in both A549 and HepG2 cells. Stress-inducible HSPA1A and HO-1 were also significantly upregulated in a dose-dependent manner. A higher level of HSPA1A and HO-1 activation by Ag NPs occurred in HepG2 cells than that in A549 cells. Compared with that of HSPA1A, Ag NPs induced a stronger increase in protein level of HO-1 in both cell lines. Significant positive correlations between protein levels of HSPA1A and HO-1 and oxidative damage were also observed. In conclusion, Ag NPs could induce oxidative stress in human cell lines. In addition to the products of oxidative stress such as MDA and 8-oxo-dG, HSPs can be used as potential biomarkers in nanotoxicity assessment, especially HO-1.