Exposure to lead (Pb) is a serious public health concern for which there is no safe level. The aim of this study was to investigate the toxicity of lead to undifferentiated and differentiated human neuroblastoma (dSH-SY5Y) cells and to evaluate gene transcription in response to sub-lethal lead exposure. Pb was applied to undifferentiated and dSH-SY5Y cells across a concentration range of 0-5 mM for 4, 6, and 24 hours, and cell viability was assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase assays. Pb induced a significant concentration- and exposure-dependent reduction in cell viability (p < 0.0001), with IC₅₀ effects at approximately 3 and 2.5 mM for undifferentiated and dSH-SY5Y cells, respectively. Pb also significantly reduced cellular bioenergetics capacity in a concentration and exposure duration manner (p < 0.0001) and resulted in elevated levels of deleterious reactive oxygen species (p < 0.0001). RNA sequencing was undertaken to analyse gene transcription in dSHSY-5Y cells in response to a 24-hour exposure to 1.25 mM Pb. Differentially expressed gene (DEG) analysis revealed significant transcriptomic alterations in dSH-SY5Y cells following Pb exposure. A total of 2963 DEGs were identified, of which 757 genes were significantly upregulated, 2206 genes were significantly downregulated, while 26,461 genes showed no significant change. From Gene Ontology analyses, biological processes were predominantly associated with immune and inflammatory responses. From Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis, upregulated DEGs were particularly enriched in immune pathways, whereas downregulated DEGs were enriched in pathways related to cellular metabolism, homeostasis, and neurodevelopment. These findings offer molecular insights into Pb-induced neurotoxicity and highlight the potential for Pb exposure to affect both the immune system and neurodevelopment.