The widespread use of the organophosphorous insecticide chlorpyrifos (CPF) over recent decades has posed major concerns about its toxicity in humans. Sub-cytotoxic concentrations of CPF and its metabolite CPF-oxon (CPO) were known to inhibit neurite outgrowth in differentiating neural cells but little was known about their ability to cause neurite retraction. The main aims of this study were to investigate the effects of CPF and CPO on the stability of neurites in pre-differentiated mouse N2a neuroblastoma and human ReNcell CX neural stem cells, and to relate toxicity to the regulation of cytoskeletal proteins involved in neural differentiation. At 3 μM, both compounds reduced the numbers of axon-like processes in pre-differentiated N2a cells, as indicated by morphometric analysis of carboxyfluorescein succinimidyl ester-labelled cells. Retraction of neurites was observed within 2 h of exposure by live cell imaging. Neurofilament disruption was detected in treated cells, by indirect immunofluorescence with anti-phosphorylated neurofilament heavy chain (pNFH) monoclonal antibody SMI34, while the microtubule network was unaffected. Western blotting analysis revealed transiently increased levels of reactivity of Ta51 after 2 h exposure but reduced levels of reactivity following 8 h treatment with both compounds, whereas reactivity of anti-total NFH or anti-tubulin were unaffected. Altered NFH phosphorylation at 2 h exposure was associated with increased activation of extracellular signal-regulated protein kinase ERK 1/2. Increased levels of phosphatase activity were observed following 8 h treatment, suggesting that organophosphate-induced neurite retraction in N2a cells is associated with early transient increases in NFH phosphorylation and ERK1/2 activation. High content analysis of immunofluorescently stained N2a cells showed that the induction of neurite retraction by both compounds was concentration-dependent. The same concentrations of CPF and CPO also caused retraction of neurites in differentiating neuronal and glial populations of human ReNcells. Using a cell ELISA technique changes observed in Western blot analysis were confirmed and found to be concentration-dependent in N2a cells. In pre-differentiated ReNcells, reduced levels of NFH phosphorylation were observed, whereas total NFH, βIII-tubulin and ERK1/2 activation were unaffected. Acetylcholinesterase assays suggested that inhibition was not required for neurite retraction but could affect the severity of such effects.