Abstract: This study aims to determine the plant-based formation of Sb-nanoparticles in vitro and in situ by single particle ICP-MS analysis. In vitro experiments showed that leaves of terrestrial yellow box Eucalyptus sp. and semi aquatic Typha sp. produced Sb-nanoparticles, with great variation in particle size ranging from 30 to 100 nm and 30 to 90 nm, respectively. A linear relationship between Sb concentration and particle size was detected for both terrestrial and semi-aquatic plants. Terrestrial plants showed a good linear model when using the inorganic oxidised form Sb(V) (antimonate) to form Sb-nanoparticles. By contrast, semi-aquatic plants showed a good linear model when using the inorganic reduced form Sb(III) (antimonite). This suggests a preferential formation of plant-based Sb-nanoparticles based on plant type and Sb forms. Preliminary in situ experiments showed that weed Urtica dioica (Stinging nettle) and native grass Austrostipa sp., growing on Sb-rich mineral waste, accumulated higher levels of Sb in various tissues parts. For instance, Stinging nettle accumulated nearly four times as much Sb in their root tissue compared to Austrostipa sp. with 3720 ug/kg and 1000 ug/kg, respectively. By contrast, above ground parts accumulated similar concentrations of Sb between plants species, 130 ug/kg and 100ug/kg, respectively. As for Sb nanoparticle formation, Austrostipa sp. produced bigger particle size in roots (118 nm) relative to shoots (83 nm); while Stinging nettle produce similar particle size between shoots and roots with 162 nm and 163 nm, respectively. Overall, the variation in particle size between in vitro and in situ methods suggests that mechanism of Sb-detoxification may play an important role in the variation of Sb-nanoparticles formation in plants. More detail testing of in situ Sb accumulation in U. dioica and Austrostipa sp. is required to determine the plant molecules and Sb forms involved in the Sb-nanoparticles formation.