Superparamagnetic iron oxide nanoparticles (SPIONs) are used in many biological applications, which necessitate intracellular targeting. Here, we investigate intracellular localization and gene expression in HeLa cells after treatment with functionalized SPIONs. Functional groups investigated included positive amino propyl silane (APS), polyethylene glycol and targeting peptides: nuclear targeting peptide (NTP) and/or cancer cell uptake promoting peptide (cRGD). Results revealed that the intracellular localization of SPIONs was strongly dependent on the surface chemistry. Nuclear targeted SPIONs functionalized with only NTP or both NTP and cRGD were mostly localized in perinuclear endosomes with a small fraction entering the nucleus. The biocompatibility of cells after treatment was also dependent on surface chemistry, where SPIONs functionalized with both NTP and cRGD exhibited a more significant reduction of cell proliferation compared to NTP or cRGD individually. Interestingly, gene expression after treatment with SPIONs was similar, regardless of the surface functionalization or intracellular localization. The results of this study showed that cellular uptake and intracellular localization predominantly depended on the surface chemistry, while gene expression exhibited a more generic response to SPION treatment.