Roads are a significant cause of habitat fragmentation and a barrier to movement of several species across the world. Terrestrial species with low mobility, low abundance and specific habitat requirements could be particularly affected by this kind of barriers concerning their movements. In the present study, we aimed to assess the influence of habitat invasion by roads on space use and movements of Cabrera voles (Microtus cabrerae), an Iberian endemism, classified as ‘near threatened’ and with small and fragmented populations. We hypothesise that: (1) individuals occupying areas more densely encroached by roads have smaller home ranges and higher intrasexual spatial overlap; (2) movement paths are i) shorter; ii) more linear; and iii) more frequent along the road in areas with more encroachment; (3) movements are shorter during higher traffic periods of the day (daylight) in those areas. We radio-tracked 16 Cabrera voles (Wildlife Materials; SOM-2018) in two habitat patches along a National road in Southern Portugal (Évora district). The patches are separated by 1.4 km, with one being highly encroached by roads (“verge” patch) and the other much less impacted by these structures (“meadow” patch), with high habitat availability despite the proximity of roads. Eight voles were radio-tracked in each patch (1 male and 7 females in “Meadow”; 5 males and 3 females in “Verge”), between 7th April and 14th June 2017. Fixes were registered at 15 min-intervals in rounds of 4 hours which covered the 24h cycle. We measured home range size (biased random bridge kernel) for all individuals, the percentage of home range overlap between females (UDOI index), path length, path linearity, and path and step direction to assess space use and movement patterns in the two habitat patches. According to our predictions, animals occupying the “verge” patch showed significantly smaller home ranges when compared with animals sampled in the “meadow” patch, although female spatial overlap was not statistically different between patches. Some of the remaining predictions were not satisfied, as there were no significant differences in path length, path linearity or step direction between patches. Nevertheless, there were differences in path length between day periods but only for the “verge” patch. The paths were shorter for the period 09-21h when compared with 05-09h in verge patch (but not in meadow patch). No events of road crossing were detected in any of the radio-tracked animals. Overall, these results provide empirical support for the idea that habitat invasion by roads may significantly impact species behavioural traits related to space use and movement patterns.