Occurrences of mountain lions (Puma concolor) in Nebraska have been steadily increasing; however, reproductive activity in mountain lions has not been documented in the state. We present the first evidence of mountain lion reproduction in Nebraska since mountain lions recolonized the state in the early 1990s, On 28 February 2007, a spotted kitten was hit by a vehicle in northwestern Nebraska; and based on body length and weight, we estimate its age at 3.9 months. On 20 December 2008, a female mountain lion and spotted kitten were photographed in the northwestern part of the state, On 9 May 2009, a female mountain lion with a juvenile was also photographed. All records were from the Pine Ridge region of Dawes County, Nebraska, Our records suggest that mountain lions are establishing a permanent population in at least one region of Nebraska.