Uterine neural connections represent a simple neural circuit to study the finest mechanisms of axon remodeling. Neurons innervating the uterus respond to physiological alterations in systemic levels of estrogen (E2) with changes in their terminal axons. Sympathetic axons disconnect when E2 rises and re-connect when E2 is low. The uterus releases diffusible molecules that exert neuronal sub-set-specific effects. Here we investigated if NRP1 and NRP2 signaling could mediate as negative regulators of sympathetic axonal growth. Functional blocking antibodies against NRP1 and NRP2 in three-dimensional co-cultures of sympathetic ganglion/myometrial explants were employed. Female rats of different hormonal conditions (low or high systemic E2) were used as tissue donors. There was no difference in axonal length and direction of outgrowth in sympathetic axons extended from ganglionic explants co-cultured with myometrial control explants (GC vs C-myo; P/D ratio = 1). However, when ganglionic explants were co-cultured with E2-myo explants axons were strongly inhibited and repelled (GE vs E2-myo; P/D ratio < 1). This effect depended on the distance between explants. Axon outgrowth inhibition by E2-myo explants was prevented by NRP2 blockade (Nrp2B GE vs E2-myoP/D ratio=1). In contrast, blocking NRP1 did not avoid the axon outgrowth inhibition exerted by E2-myometrium. Together, our data demonstrate the exclusive requirement of NRP2 in E2-induced impairment of sympathetic axon outgrowth.