Gold nanorods (GNRs) are of interest in many biological applications, including drug delivery and biomedical imaging due to their unique optical properties and the versatility for surface modification. For use in such applications, it is important to understand the effect of surface chemistry on the toxicological effects of GNRs to cells. In the current study, GNR stabilized with cetyltrimethylammonium bromide (GNR-CTAB) and GNR functionalized via a ligand exchange method with either thiolated polyethylene glycol (PEG) 5000 or mercaptohexadecanoic acid (MHDA) were investigated for their stability in biological media and toxicological effects to HaCat cells. Cell viability assays demonstrated minimal toxicity of GNR-PEG and GNR-MHDA and high toxicity of GNR-CTAB, which was found to be due to the inherent toxicity of the cationic surfactant to cells. Due to this high level of toxicity, further studies focused only on GNR-MHDA and GNR-PEG. Cell uptake studies indicated relatively low uptake for GNR-PEG and high uptake for GNR-MHDA. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine effects at the genetic level and showed that GNR-PEG did not induce any effects, while GNR-MHDA influenced several of the genes investigated. Based on this information GNR-PEG is ideal for low cell uptake and high stability in biological media, while GNR-MHDA may be desirable for increased uptake of the GNRs into cells. However, further investigation into the potential effect of GNR-MHDA on gene expression is required.