Demyelination mouse models are often used to investigate the mechanisms of demyelination and subsequent remyelination in diseases characterized by white matter injury. Toxin-induced focal demyelinating lesion in the mammalian central nervous system (CNS) is a common and useful demyelination animal model. Injection of L-α-lysophosphatidylcholine (LPC) into the CNS causes focal demyelination and neurological deficits corresponding to the demyelination site. The neurological deficits partially recover after injury, which is correlated with spontaneous remyelination. Here, we describe the protocol for LPC injection into the mouse spinal cord, which can be completed within 30 minutes. LPC injection into the dorsal column of the spinal cord at the lower thoracic level in mice causes motor dysfunction followed by partial recovery and spontaneous remyelination. We also describe the protocols for two behavioral tests to quantify changes in motor function post-injury and after recovery.