Secondary axonal loss contributes to the persistent functional disability following trauma. Consequently, preserving axons following spinal cord injury (SCI) is a major therapeutic goal to improve neurological outcome; however, the complex molecular mechanisms that mediate secondary axonal degeneration remain unclear. We previously showed that IP₃R-mediated Ca²⁺ release contributes to axonal dieback and axonal loss following an ex vivo laser-induced SCI. Nevertheless, targeting IP₃R in a clinically relevant in vivo model of SCI and determining its contribution to secondary axonal degeneration has yet to be explored. Here we used intravital two-photon excitation microscopy to assess the role of IP₃R in secondary axonal degeneration in real-time after a contusive-SCI in vivo. To visualize Ca²⁺ changes specifically in spinal axons over time, adult 6–8 week-old triple transgenic Avil-Cre:Ai9:Ai95 (sensory neuron-specific expression of tdTomato and the genetic calcium indicator GCaMP6f) mice were subjected to a mild (30 kdyn) T12 contusive-SCI and received delayed treatment with the IP₃R blocker 2-APB (100 μM, intrathecal delivery at 3, and 24 h following injury) or vehicle control. To determine the IP₃R subtype involved, we knocked-down IP₃R3 using capped phosphodiester oligonucleotides. Delayed treatment with 2-APB significantly reduced axonal spheroids, increased axonal survival, and reduced intra-axonal Ca²⁺ accumulation within dorsal column axons at 24 h following SCI in vivo. Additionally, knockdown of IP₃R3 yielded increased axon survival 24 h post-SCI. These results suggest that IP₃R-mediated Ca²⁺ release contributes to secondary axonal degeneration in vivo following SCI.