Much of the concerns regarding engineered nanoparticle (NP) toxicity are based on knowledge from previous studies on particles in ambient air or occupational situations. E.g., the effects of exposure to silica dust particles have been studied intensely due to the carcinogenicity of crystalline silica. However, the increasing usage of engineered amorphous silica NPs has emphasized the need for further mechanistic insight to predict the consequences of exposure to the amorphous type of silica NPs. The present study focused on the in vitro biological effects following exposure to well-dispersed, BSA-stabilized, amorphous silica NPs whereas unmodified silica NPs where included for reasons of comparison. The cytotoxicity of the silica NPs was investigated in six different cell lines (A549, THP-1, CaCo-2, ASB-XIV, J-774A.1, and Colon-26) selected to explore the significance of organ and species sensitivity in vitro. Viability data demonstrated that macrophages were most sensitive to silica NP and interestingly, murine cell lines were generally found to be more sensitive than comparable human cell lines. Further studies were conducted in the human epithelial lung cell line, A549, to explore the molecular mechanism of silica toxicity. Generation of reactive oxygen species, one of the proposed toxicological mechanisms of NPs, was investigated in A549 cells by the dichlorofluorescin (DCF) assay to be significantly induced at NP concentrations above 113 μg/mL. However, induction of oxidative stress related pathways was not found after silica NP exposure for 24 h in gene array studies conducted in A549 cells at a relatively low NP concentration (EC20). Up-regulated genes (more than 2-fold) were primarily related to lipid metabolism and biosynthesis whereas down-regulated genes included several processes such as transcription, cell junction, extra cellular matrix (ECM)-receptor interaction and others. Thus, gene expression data proposes that several cellular processes other than oxidative stress could be affected by exposure to silica NPs.