Chlorpyrifos (CPF) and CPF-oxon (CPO) are known to inhibit neurite outgrowth but little is known about their ability to induce neurite retraction in differentiating neuronal cells. The aims of this study were to determine the ability of these compounds to destabilize neurites and to identify the key molecular events involved. N2a cells were induced to differentiate for 20 h before exposure to CPF or CPO for 2–8 h. Fixed cell monolayers labeled with carboxyfluorescein succinimidyl ester or immunofluorescently stained with antibodies to tubulin (B512) or phosphorylated neurofilament heavy chain (Ta51) showed time- and concentration-dependent reductions in numbers and length of axon-like processes compared to the control, respectively, retraction of neurites being observed within 2 h of exposure by live cell imaging. Neurofilament disruption was also observed in treated cells stained by indirect immunofluorescence with anti-phosphorylated neurofilament heavy chain (NFH) monoclonal antibody SMI34, while the microtubule network was unaffected. Western blotting analysis revealed transiently increased levels of reactivity of Ta51 after 2 h exposure and reduced levels of reactivity of the same antibody following 8 h treatment with both compounds, whereas reactivity with antibodies to anti-total NFH or anti-tubulin was not affected. The alteration in NFH phosphorylation at 2 h exposure was associated with increased activation of extracellular signal-regulated protein kinase ERK 1/2. However, increased levels of phosphatase activity were observed following 8 h exposure. These findings suggest for the first time that organophosphorothionate pesticide-induced neurite retraction in N2a cells is associated with transient increases in NFH phosphorylation and ERK1/2 activation.