We have developed a pyramidotomy model in mice to lesion the corticospinal tract at the level of the brainstem pyramidal tract, and evaluated the resultant impairments in motor function in a series of behavioural tests. Adult C57BL/6 mice received a unilateral pyramidotomy and a control group of mice underwent sham surgery. We studied the effects of this lesion on forepaw function using five behavioural paradigms, some of which have been widely used in rat studies but have not been fully explored in mice. The tests used were: a rearing test, which assesses forepaw use for weight support during spontaneous vertical exploration of a cylinder; a grid walking test, which assesses the ability to accurately place the forepaws during exploration of an elevated grid; a tape-removal test, which measures both sensory and motor function of the forepaw; a CatWalk automated gait analysis, which provides a number of quantitative measures including stride length and stride width during locomotion; and a staircase reaching task, which assesses skilled independent forepaw use. All tests revealed lesion effects on forepaw function with the tape removal, grid walking, rearing and CatWalk tests demonstrating robust effects throughout the testing period. The development of a pyramidotomy lesion model in mice, together with behavioural tests which can reliably measure functional impairments, will provide a valuable tool for assessing therapeutic strategies to promote regeneration and plasticity.