Previous research has repeatedly associated the expansion of the Vγ9Vδ2 T cell population during Plasmodium falciparum infection with malaria protection, however the cellular mechanisms behind this association are not fully characterised. We propose that increased cytotoxic function during Pf infection, is one mechanism by which Vγ9Vδ2 T cells confer malaria protection. Vγ9Vδ2 T cells have innate cytotoxic potential and can execute direct cytotoxic killing of blood-stage Pf in vitro. Clinical studies in malaria-endemic children have also correlated higher CD16+ Vγ9Vδ2 T cell frequencies and increasing age with clearance of Pf infection. In our study we aimed to characterise the emergence of this cytotoxic phenotype during primary Pf infection and investigate age-related differences in the cytotoxic potential of Vγ9Vδ2 T cells. Firstly, we investigated the activation of cytotoxic Vγ9Vδ2 T cells during a Controlled Human Malaria Infection trial (CHMI) of malaria-naive adults. In this CHMI, participants were intravenously inoculated with 2800 intact Pf parasitised red blood cells, and treated with an anti-malarial drug at 8-days post-infection. Peripheral blood was drawn from participants (n = 8) at 0-, 8-, 15- and 42-days post-infection. Surface and intracellular staining of cryopreserved PBMCs revealed the activation of a highly cytotoxic Vγ9Vδ2 T cell at 15-days post-infection. The expanded, cytotoxic Vγ9Vδ2 T cell population had increased frequencies of CD16 and CD56 surface expression, as well as, increased intracellular production of perforin, granzyme-B and granulysin. Next, we performed ex vivo and in vitro phenotyping of PBMCs from malaria-naive children, to delineate associations of age and Pf exposure with the expansion of cytotoxic Vγ9Vδ2 T cells. We correlated increased age with decreased CD16+ Vγ9Vδ2 T cell frequency and reduced intracellular production of granzyme-B after stimulation with blood-stage Pf. Overall, we showed the expansion and activation of cytotoxic Vγ9Vδ2 T cells 15-days after primary Pf infection of malaria-naive adults in vivo, and decreased cytotoxic Vγ9Vδ2 T cell frequency with age in malaria-naive children. Further analysis aims to investigate the association of prior cytomegalovirus infection and the augmentation of the cytotoxic Vγ9Vδ2T cell phenotype within these cohorts.