In response to growing concerns about public health risks of antibiotic resistance from food production, a range of regulations have been implemented on agricultural antibiotic use worldwide. Although the Danish yellow card initiative, antibiotic quantitative restriction, is considered as the most sophisticated latest addition, no rigorous empirical research has been conducted yet. This article provides the first empirical estimates of the effect of the yellow card initiative on the economic performance of farmers. The results show that the initiative has reduced gross profit and increased operating expenses of farmers. As such, farmers have spent more labour hours and costs performing biosecurity, increased spending on veterinary medical services and feed, which might have driven the reduction in profit.