Gene regulatory DNA regions are essential for controlling gene expression and they contain a significant proportion of disease-associated variants. Understanding variation within gene regulatory regions may facilitate understanding of disease progression or the development of stratified medicines. The CB1 receptor is involved in controlling many physiological processes such as appetite and reward signalling and has been investigated as a drug target. Differences in response and side effects, however, may indicate that genetic variation within the CB1 receptor gene (CNR1), may need to be investigated to better predict disease and drug efficacy. As disease-associated polymorphisms have been identified in non-coding sequences of CNR1, the aim was to investigate the role of CNR1 regulatory elements and how genetic and epigenetic variation influence their activity. This study identified that the CNR1 promoter is active in primary cell cultures generated from the hypothalamus and dorsal root ganglia (DRGs) of rats and is highly susceptible to CpG methylation in DRGs. Additionally, a highly conserved intronic sequence, ECR1, was disrupted in mice using the CRISPR/Cas9 system. Disruption of ECR1 led to reduced CNR1 expression in the hippocampus but not in the hypothalamus as well as reduced CB1 agonistinduced hypothermia, a reduction in ethanol intake and altered anxiety-like behaviour. The major allele of a SNP within ECR1, permitted CNR1 promoter activity and the minor allele significantly repressed CNR1 promoter activity in hypothalamic and DRG cell cultures. In DRGs, allelic variation within ECR1 altered the response of the CNR1 promoter to the CB1 receptor antagonist/inverse agonist, rimonabant. In conclusion, this study indicates that ECR1 is a tissue specific enhancer and may be involved in regulating physiological processes, and, genetic variation in ECR1 can alter its control of the CNR1 promoter. Overall, this study elucidates possible CNR1 gene regulatory mechanisms, and how genetic and epigenetic variation influences gene regulation.