Groundwater within the outcrop area of the Lincolnshire Limestone aquifer in eastern England is characterized by high (>50 mg l−1) nitrate concentrations, whereas in the confined zone c. 15 km further east, nitrate concentrations are low (<0.5 mg l−1) or undetected. The decrease in nitrate concentrations is associated with an increase in the 15N/14N ratio (or δ15N) of the residual nitrate, a feature characteristic of bacterially mediated denitrification. The calculated isotopic enrichment factor for the denitrification process of −1.8‰ is low when compared with literature reports of laboratory experiments and previous field investigations. Isotope evolution modelling shows that the low enrichment factor is related to complex mixing processes occurring within the aquifer as a result of the interactions between fissure flow, matrix diffusion and mixing with low-nitrate groundwater in the confined zone. The natural attenuation of nitrate is proved to occur and is further investigated with a finite-element reactive transport fractured media model influenced by denitrification. The model simulations show that oxygen and nitrate concentrations decrease with distance from the centre of a fracture to values of 0.2 mg l−1 and 1.0 mg l−1, respectively, at a distance of 4 cm from the fracture walls, in agreement with field evidence. The bacterial consumption rates of oxygen and nitrate mass can be calculated by comparison of reactive and non-reactive simulations and are found to be 4.2 mg O2 m−3 day−1 and 48.2 mg NO3 m−3 day−1. Assuming heterotrophic denitrification, the organic carbon substrate utilization rates are calculated to equal 0.13 mg l−1 day−1 at outcrop (equivalent to the left-hand boundary of the model domain) and 0.07 mg l−1 day−1 at the right-hand boundary of the domain at a distance of 6.5 km. From a consideration of the percentage of available organic carbon contained in the limestone, and with a consumption rate of 0.13 mg l−1 day−1 at outcrop, the whole reservoir of organic carbon in the model domain would be utilized in 209 years, thus suggesting an eventual limit on denitrification in the confined aquifer.