Mutants of brain-derived neurotrophic factor (BDNF) are associated with obesity. However, the regulatory mechanism of BDNF expression is still unclear. We developed a novel mutant mice line named Timo (transgenic insertional mutants with obesity) in which a potential regulatory locus of Bdnf was disrupted by transgene insertion. The insertion site was identified and lies 857 kb upstream of the Bdnf gene. The disrupted genomic locus is conserved across mouse, rat, dog and human genome and contains several highly conserved elements that are able to up-regulate reporter gene expression in vitro. Along with down-regulation of BDNF to about 30% of wild-type animals, Timo/Timo mice exhibited increased body weight and fat content with hepatic steatosis and elevated serum levels of leptin, cholesterol and LDL-cholesterol. These mutant mice also showed obesity-independent insulin resistance, hyperinsulinaemia, impaired glucose tolerance, age-dependent hyperglycemia and shortened life span. Molecular and phenotype analysis of Timo/Timo mice indicated the existence of a genome locus, lying 857 kb upstream of the Bdnf gene, that regulates BDNF expression, body weight and glucose homeostasis.