Pest control is a global issue for agriculture, health, biodiversity conservation and economy. Anticoagulant rodenticides are used over large areas to control rodent pests and can cause widespread poisoning of nontarget wildlife. In France, bromadiolone is the only pesticide authorized to control the water vole Arvicola terrestris Scherman, in grasslands. Since 2001, legislation has been in place to replace curative treatments by preventive ones and limit the quantity of rodenticide used. As the legislation took effect over time, the impact on red fox Vulpes vulpes populations was monitored. Fox populations and bromadiolone treatments were monitored in the Doubs Department (5000 km² area), France. Fox counts were carried out during spring, and vole control was primarily conducted in autumn. Relative fox densities (Kilometric Abundance Index: KAI) obtained per commune for year n (2004-2009) were related to treatments achieved during year n−1 (2003-2008). Treatments from year n−2 were used to investigate possible delayed responses in fox populations. Kilometric Abundance Index of foxes was significantly related to treatment intensities in years n−1 and n−2. The impact was greatest in a large area (>1000 km²), where intensive treatments were achieved in 2003. Fox KAI generally remained dramatically low in this area until 2005, after which a partial recovery was observed. The same area was treated again from 2006 to 2008 but with only half the amount of bait per hectare that was used in 2003. These treatments were followed by a moderate decrease in fox populations. Synthesis and applications: We have established, for the first time on a regional scale, the negative impact of a rodenticide on fox populations. We have shown that a shift to preventive treatments with reduced anticoagulant rodenticide use is less harmful to fox populations. However, to approach a zero impact, treatments should be reduced further by limitation of bait quantities authorized per hectare and per commune and using alternative methods to chemical control. Long-term monitoring of wildlife populations using index methods can provide valuable information about the adverse effects of pesticides; therefore, we recommend their inclusion in the assessment of pest management practices.