Estrogen is a neurosteroid with protective and regenerative properties. Nerve growth factor differentiated PC12 neuronal cells have been shown to express both nuclear estrogen receptor ER alpha (genomic) and membrane estrogen receptor GPER (non-genomic), thus our aim is to differentiate which cellular pathways are employed by estrogen in neuritogenesis regulation. Here we treated such cells with 17-beta-Estradiol (E2) in a series of short and long incubation times. E2 treatments (10-6M) were compared with plasma membranous G protein-coupled estrogen receptor agonist G1 (10-6M) and antagonist G15 (10-9M) to differentiate slow from rapid pathways. Neural scaffolding protein Piccolo was detected with immunocytochemistry, Western blots and OPD-assays to analyse the influence of estrogen in neurite formations and interactions. We used live cell imaging to analyse the microanatomy of neuronal cells, with and without estrogen treatments. Neuritogenesis gradually increased as the time course progressed for E2 treatments, which supports the established slow genomic transcription theory of estrogen. Piccolo aggregates observed at neurite endings after short incubation with G1 signifies that a rapid membranous response to estrogen is possible in neurite formations. Both western blots and OPD-assays manifested these observations quantitatively. Live cell imaging data indicated that the estrogen effect and uptake on neuronal cells can be modulated with G1 and G15. Our evidence explains that estrogen has the ability to upregulate neuritogenesis not merely via the classical genomic but also via the rapid non-genomic pathway and can be applied to elucidate the estrogen dependent neuroprotective effect in oxidative stress.