Mesenchymal stem cells (MSCs) have the potential for self-renewal and multipotential differentiation to regenerate damaged tissues or recover functional absence in diseases. Superparamagnetic iron oxide nanoparticles (SPIONs) are used as contrast agents in magnetic resonance imaging (MRI) for labeling cells in vitro and for tracking SPION-labeled cells after transplantation in vivo. Human amniotic membrane-derived mesenchymal stem cells (hAM-dMSCs) have the capacity for neuron-like differentiation that could be used to cure central nervous system (CNS) diseases. The study investigated the impacts of cytotoxicity of SPIONs on neuron-like differentiation of hAM-dMSCs in both single (1×) and multiple (4×) SPIONs-labeled methods. hAM-dMSCs could be efficiently labeled at safe concentrations of SPIONs (≤14 μg/ml) without significantly affecting their viability (>80% after a MTT assay), special surface antigens (CD29, CD44, CD90, CD105 through flow cytometry), and neuron-like differentiation (nestin and neuron-specific enolase through immunocytochemistry and reverse transcription polymerase chain reaction). Compared with multiple (4×) SPION-labeled methods, a single (1×) SPION-labeled method avoided multiple SPION-labeled hAM-dMSCs and minimized the impact of cytotoxicity of SPIONs on neuron-like differentiation of hAM-dMSCs. Under safe concentrations of SPIONs, a single (1×) SPION-labeled method provided appropriate viability for SPIONs-labeled hAM-dMSCs and facilitated the MRI evaluation of hAM-dMSCs after transplantation.