The increasing application of nanodiamond and its derivatives in many industries and healthcare products warrants a critical and urgent need to elucidate potential adverse effects. This study evaluated the cell viability and gene expression patterns of human lung epithelial cell line (Calu-3) exposed to nanodiamond (ND) and 4 surface functionalized nanodiamonds (SF-NDs): glycine-ND (Gly-ND); glucose-ND (Glu-ND); fluorinated-ND (F-ND) and ethylenediamine-ND (EDA-ND). The ND and SF-NDs were characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), dynamic light scattering (DLS) and attenuated total reflectance Fourier transformed infrared (ATR-FTIR) spectrum. The cytotoxicity of ND and SF-NDs was evaluated using MTS assay. Gene expression profiles were generated using microarray analysis for Calu-3 cells exposed to ND. The results of XRD and ATR-FTIR spectra provided evidence for successful functionalization of ND. The morphological and particle size analysis using SEM and DLS revealed that ND and SF-NDs form agglomeration. The cytotoxicity study data showed that ND and SF-NDs exhibit concentration dependent material-specific toxicity with the general trend for biocompatibility: Gly-ND > Glu-ND > ND > F-ND > EDA-ND. Microarray analysis indicated a subtle cellular response to ND with few genes affected more than 2-fold up or down. However, some gene expressions such as NAD(P)H dehydrogenase (NQO1) showed up-regulation while a gene associated with the metastasis of lung adenocarcinoma transcript 1 (MALAT1) was down-regulated. In conclusion, at the concentrations of < 100 µg/mL, ND and SF-NDs appeared to be safe to human lung epithelial cells in vitro after 24 hrs exposure.