Aims <p>The aim of this work was to shed light on the still debated mechanisms of Ni tolerance in facultative serpentinophytes and their photosynthetic performance in presence of Ni. To this end, three non-serpentine and three serpentine accessions of <i>Silene paradoxa</i> L. collected in Tuscany were compared.</p> Methods <p>Plants were grown in hydroponics with NiSO₄ treatments and root elongation, Ni accumulation, and photosynthetic performance evaluated.</p> Results <p>The presence of Ni reduced plant growth and led to different metal accumulation patterns between the two plant groups. Tolerance and accumulation parameters suggest that serpentine adaptation required enhanced Ni tolerance in <i>S. paradoxa</i>, likely driven by the selective pressure imposed by high metal concentration in the substrate. Ni tolerance was linked to the ability to limit the metal entry in the root symplast rather than being associated with high tissue tolerance or shoot exclusion. Leaf gas exchange data and chlorophyll fluorescence data revealed that Ni excess differentially affected stomatal opening in the two plant groups, with cascading effects on assimilation rates and, consequently, growth. Ni excess, rather than affecting leaf photochemistry per se, differentially affected stomatal opening, with declines in stomatal conductance observed only in the non-serpentine accession. This indicates that greater Ni tolerance in the serpentine accession was linked with improved water relations rather than enhanced Ni tissue tolerance at the photosynthetic level.</p> Conclusions <p>Overall, all results indicate that metal exclusion is the main mechanism of Ni tolerance in <i>S. paradoxa</i>, thus reducing the need for more energy-demanding strategies of internal tolerance.</p>