Use of exclusion fencing is an effective method to reduce moose-vehicle collisions, and exclusion fences are commonly erected along Swedish highways. However, exclusion fences may pose a threat to the viability of wildlife populations because they serve as barriers to individual movements and may limit accessibility to resources. Various types of wildlife crossings intended to reduce road-kills and increase habitat connectivity across fenced highways have been constructed throughout the world. However, few studies have evaluated the effectiveness of these crossing structures with respect to movements before, during and after construction of highways and exclusion fencing. We studied movements of 24 GPS-collared moose Alces alces before, during and after an existing two-lane road was reconstructed to a fenced four-lane highway with three wildlife crossings designed for moose. We recorded 135 movements across the highway during 8,830 moose-monitoring days. Of these, 47 occurred before the construction began, 76 occurred during the construction, and 12 occurred after the highway was fenced. All movements registered after the fencing occurred across two of the three wildlife crossings. The average number of highway crossings per moose-day decreased by 67-89% after fencing. The number of moose-vehicle collisions decreased after the exclusion fencing, but the fenced highway served as a barrier to moose movements even though three wildlife crossings were created. Thus, exclusion fencing may reduce moose mortality and provide safer conditions for automobile travellers, but the fencing may have a negative impact on moose accessibility to resources, gene flow and recolonisation rates.