Targeted deletion of the neuronal basic helix–loop–helix transcription factor Nhlh2 results in adult-onset obesity in mice. Measurement of body weight and body composition in animals aged 3–25 weeks indicates that while male and female Nhlh2 knockout (N2KO) animals both show adult-onset obesity, the time frame for development of obesity is different, with females becoming obese by 7 weeks of age and males becoming obese by 10 weeks of age. Heterozygous (HET) animals also become obese but with a slower onset, indicating a dosage effect for the activity of the Nhlh2 transcription factor. Food intake, body temperature, and voluntary activity were measured in both preobese and obese N2KO, HET, and wild-type (WT) animals to determine which factors contributed to weight gain. While increased food intake and decreased body temperature were found in older obese N2KO animals, only reduced physical activity preceded the onset of obesity in N2KO mice. N2KO animals had no deficit in either circadian rhythm or balance and motor control, indicating that reduced voluntary activity is the result of a behavioral change. These data demonstrate a role for the Nhlh2 transcription factor in controlling genes important to energy expenditure, and more specifically voluntary physical activity of animals.