This paper describes the neuroprotective and axon regenerative effects of siRTP801, which is a translatable small-interfering RNA (siRNA) targeting mTOR negative regulator; RTP801, through in-vivo and in-vitro models. In-vivo studies involved an optic nerve crush rat model with intravitreal injections of siRTP801 on days zero, eight and 16 post crush. Intravitreal injection of siEGFP served as a model in the contralateral eye as well as a group of uninjured animals as intact controls. At day 24 tissue was harvested for immunohistochemistry and analysis of retinal ganglion cell (RGC) survival, axonal regeneration, glial activation, RTP801 expression and mTOR activity by detecting ps6 expression; a downstream indicator of mTOR activity. In-vitro studies involved mixed retinal cell cultures that were lipofected with siRTP801 and siEGFP and treated with rapamycin which are then tested for NGF, NT-3 and BDNF levels, number of surviving βIII tubulin+ RGC and number of GFAP+ activated glia. They found treatment with siRTP801 promoted increase in Brn3a+ RGC survival at 24 days post optic nerve crush, supporting its neuroprotective role. It also promoted longer growth of regenerating axons. In-vitro studies showed that this was not initiated in the absence of activated retinal glia. Further work showed siRTP801 did not promote glial proliferation and only reduced RTP801 expression. However, GFAP+ activated glia were present in retinal cultures five days post optic nerve crush, indicating this to be an injury response. Furthermore, neurotrophin titres were increased; NGF in particular, for retinal cultures post crush but not in cultures where glia were absent. This suggests that siRTP801 potentiates neurotrophin production but not without the initiation of it by optic nerve crush injury. This study elegantly supports RTP801 as a neuroprotective and axon regenerative treatment and outlines a mechanism of action via potentiation of PI3K pathway signaling in RGC causing paracrine secretion of neurotrophins by activated glia.