This study investigated how supplementation of the diet of dairy cows with trace minerals (zinc, manganese, copper and cobalt) affected kinematic gait parameters and lactation performance. Eight Holstein cows were divided into two groups, with each group receiving a different dietary treatment (control diet, or control diet supplemented with trace minerals) in a two-period crossover design. Kinematic gait parameters were calculated by using image analysis software. Compared to cows fed the control diet, cows that received the trace mineral-supplemented diet exhibited significantly increased walking and stepping rates, and had a shorter stance duration. Feed intake and milk production increased in cows fed the trace mineral-supplemented diet compared with control groups. The plasma manganese concentration was not different in control and experimental cows. In contrast, cobalt was only detected in the plasma of cows fed the supplemented diet. These results provide the first evidence that trace mineral supplementation of the diet of dairy cows affects locomotion, and that the associated gait changes can be detected by using kinematic gait analysis. Moreover, trace mineral supplementation improved milk production and only minimally altered blood and physiological parameters in dairy cows.