Two-photon microscopy enables high-resolution in vivo imaging of cellular morphology and activity, in particular of population activity in complex neuronal circuits. While two-photon imaging has been extensively used in a variety of brain regions in different species, in vivo application to the vertebrate spinal cord has lagged behind and only recently became feasible by adapting and refining the experimental preparations. A major experimental challenge for spinal cord imaging is adequate control of tissue movement, which meanwhile can be achieved by various means. One set of studies monitored structural dynamics of neuronal and glial cellular components in living animals using transgenic mice with specific expression of fluorescent proteins. Other studies employed in vivo calcium imaging for functional measurements of sensory-evoked responses in individual neurons of the dorsal horn circuitry, which at present is the only part of rodent spinal cord grey matter accessible for in vivo imaging. In a parallel approach, several research groups have applied two-photon imaging to sensorimotor circuits in the isolated spinal cord (in vitro) to provide complementary information and valuable new perspectives on the function of specific interneuron types in locomotor-related networks. In this review we summarize recent results from these types of high-resolution two-photon imaging studies in the spinal cord and provide experimental perspectives for improving and extending this approach in future applications.