Peatlands dominate the landscape (~50% of total area) in the Athabasca Oil Sands Region (AOSR) in northern Alberta, and as such, are susceptible to hydrological disturbance from oil sands mining and exploration. In this study, we explore the hydrochemical response of a disturbed fen (Firebag Fen) in the AOSR following the removal of a temporary access road that was operational from 2007 to 2013. Prior to road removal flow was impeded at Firebag Fen, evidenced by inundation on the up-gradient side, and a lower water table on the down-gradient side of the road. Reclamation included the road removal of the ~0.5 m thick road, including 0.15 m of clay, 0.3 m of mud and organics, and 0.05 m of gravel, which left a surficial depression following removal. Peat samples (n = 139) collected immediately following road removal demonstrated significantly greater bulk densities on the down-gradient side relative to the up-gradient side of the fen, and in the road-removed area relative to up- and down-gradient areas. These combined changes decreased the average hydraulic conductivity of the fen by ~79%, which reduced water discharge across the site. Another peat sampling campaign in 2016 (n = 48), three years after removal, showed a significant decrease in bulk density where the road was removed, to within the range of values observed at areas up and down-gradient of the road. These changes increased the average hydraulic conductivity of the entire system by 64%, compared to that which was measured in 2013, with water discharge 9 and 12% higher than in 2013, during characteristically dry and wet periods, respectively. Some changes to the water chemistry (n = 24–28 for each sampling date) of the fen were also detected but patterns were different among parameters measured. For example, median concentrations on the down-gradient side of the road were 1651% (NO3–), 280% (SO42−) and 135% (Cl−) higher relative to the up-gradient side of the road. Over time following road removal, differences were reduced to 100% for nitrate, 192% for sulphate, 108% for chloride, while other parameters remained relatively unchanged (Ca2+, Mg2+, K−). Continued peat rebound (thus increasing hydraulic conductivity) may occur over time; however, elucidating this would require long-term monitoring. It is concluded that despite the impacts that were caused to the site, the natural hydrologic regime of the fen was able to operate following removal. Removal and reclamation should be considered as a worthwhile venture for roads that extend through fens and are no longer in use.