The transcription factor STAT3 has been implicated in axon regeneration. Here we investigate a role for STAT3 in sympathetic nerve sprouting after myocardial infarction (MI) — a common injury in humans. We show that NGF stimulates serine phosphorylation (S727) of STAT3 in sympathetic neurons via ERK1/2, in contrast to cytokine phosphorylation of Y705. Maximal sympathetic axon regeneration in vitro requires phosphorylation of both S727 and Y705. Furthermore, cytokine signaling is necessary for NGF-induced sympathetic nerve sprouting in the heart after MI. Transfection studies in neurons lacking STAT3 suggest two independent pools of STAT3, phosphorylated on either S727 or Y705, that regulate sympathetic regeneration via both transcriptional and non-transcriptional means. Additional data identify STAT3-microtubule interactions that may complement the well-characterized role of STAT3 stimulating regeneration associated genes. These data show that STAT3 is critical for sympathetic axon regeneration in vitro and in vivo, and identify a novel non-transcriptional mode of action.