Abstract: The spinal cord links the brain and the peripheral nervous system and has important sensory and motor functions. Impairments in the spinal cord occur in different diseases, such as spinal cord injury, multiple sclerosis, pain, motor neuron diseases, and neurodegenerative diseases. Imaging of the spinal cord has been challenging, partly due to its small size and deep anatomical location. Additionally, in an animal model, motion artifacts further influence the in vivo imaging quality of the spinal cord. Recent advances have pushed boundaries for in vivo imaging in living animals (even behaving animals). This includes high-resolution optical intravital imaging, Raman spectroscopy, mesoscopic resolution optoacoustic imaging, positron emission tomography (PET), magnetic resonance imaging (MRI), and functional ultrasound imaging (Figure 1). In addition, new genetic circuit tracing tools have been combined with in vivo imaging tools for small animal imaging. This helps our understanding of the physiology and pathology of the spinal cord at cellular, molecular, and circuit levels. Additionally, in vivo imaging of the spinal cord has also enabled the evaluation of the efficacy of both pharmacological and nonpharmacological treatments, and monitoring of the recovery progress. In this perspective, we discuss recent advancements in in vivo spinal cord imaging in animal models, as well as challenges and future outlooks.