<p>Urbanization is an important selection pressure for natural populations. Among its consequences, urban development involves habitat fragmentation and road construction. Although the effects of roads on wildlife survival and reproduction have been evaluated, the additional immediate effects of road construction, particularly on small mammals, remain poorly explored. This study aimed to assess the impact of road construction on physiological condition (stress and nutritional profile) and anxiety-like behavior of male subterranean rodents Talas tuco-tuco (<i>Ctenomys talarum</i>). Individuals were captured at the edges of Provincial Route 11 in Buenos Aires province, Argentina (“road site”), and in the same areas where road construction activities were being carried out (“construction site”). Immediately after capture, a blood sample was collected to quantify the following physiological parameters: hematocrit, blood glucose, neutrophil: lymphocyte ratio (N: L), cortisol, triglycerides, albumin, and total protein levels. Once in the laboratory, the anxiety-like behavior was evaluated using two behavioral tests: the open field (OF) and the elevated plus maze (EPM). Individuals from the “construction site” presented higher cortisol levels and N: L ratio, whereas individuals from the “road site” showed higher hematocrit, triglycerides, and total plasma proteins. No effects of the site were observed on behavioral parameters. Our results demonstrate that road construction activity impacts the physiological condition of male <i>C. talarum</i> already subjected to road disturbance, although these effects did not translate into observable behavioral changes. Future studies are needed to understand the long-term implications of natural and anthropogenic drivers of change, and their interactions, on wildlife biology.</p>