Globally rare serpentine barren ecosystems support unique communities, but in the Mid-Atlantic region of the United States, these systems are threatened by forest encroachment in the absence of disturbance. Serpentine barrens are commonly high-stress environments with low water and nutrient availability, as well as high heavy metal content. Forest encroachment into barrens is often preceded by prolific growth of the vine, Smilax rotundifolia L. To better understand how S. rotundifolia is able to colonize these inhospitable environments, the water relations of this species were examined along a gradient from serpentine barren to encroaching forest. These data were compared to the water relations of dominant C₄ grasses in the barrens. Diurnal measurements of stomatal conductance and leaf water potential were made in June, July, and August 2012 at three study sites in southeastern Pennsylvania. Predawn water potential was often highest for S. rotundifolia growing in serpentine barrens, indicating higher soil moisture availability in these areas relative to the encroaching forest. However, no evidence of water stress in diurnal measurements of stomatal conductance or water potential was detected for S. rotundifolia along the barren to forest gradient. Similarly, neither of the C₄ grasses exhibited signs of water stress, and both grasses maintained high stomatal conductance in the afternoon, relative to S. rotundifolia. The C₄ grasses are likely to have a competitive advantage over S. rotundifolia, in terms of their ability to maximize carbon gain over the course of a day and growing season, but these species are liable to suffer if they become overtopped by S. rotundifolia. Smilax rotundifolia was not limited by water availability during the 2012 growing season, calling into question the common assertion that serpentine barrens impose plant moisture stress. Given its height advantage, S. rotundifolia is likely to effectively compete with serpentine barren vegetation in the absence of disturbance or active management.