Selective pressures acting on plant life histories can drive extreme specialization. One example of such specialization is the evolution of dioecious breeding systems. Evolutionary and ecological theory posits that dioecy may subject male and female individuals to different selective pressures and result in unique sex-mediated adaptive traits related to resource allocation and ecophysiology. Cycads are the earliest diverging lineage of seed plants with strict dioecy, yet we know almost nothing about the ecology and physiology of this group. Especially limited is our understanding of potential sex-specific differences and how such differences may influence species ecology. Here we examine the ecophysiology of male and female cycads to understand better, the role that dioecy plays in this group. We evaluated sex-specific differences in ecophysiological traits and resource acquisition in five species. Specifically, we compared photosynthetic physiology, nitrogen and carbon content, isotope discrimination (δ15N and δ13C), and stomatal density. In some cycads, (i) males and females have similar investments in leaf nitrogen but females exhibit greater incorporation of nitrogen from nitrogen-fixing soil bacteria, (ii) males display higher photosynthetic capacity but females show decreased [corrected] water-use efficiency, and (iii) males have higher stomatal conductance but similar stomatal density to females. This study is the first to examine the ecophysiological differences that have evolved in the oldest dioecious lineage of seed-bearing plants. Our results show unexpected differences in photosynthetic physiology and highlight the co-evolution with nitrogen fixing soil bacteria as a potential new key player in an old lineage.