Apparent absorption of copper (Cu) in ruminants is low, with between 0.02 and 0.07 g/g Cu from sources such as Cu oxide (CuO) being absorbed resulting in high levels of excretion. Improving bioavailability of Cu could reduce the dose required to maintain Cu status, particularly in the presence of dietary antagonists to Cu absorption such as sulfur (S) and molybdenum (Mo). One way in which bioavailability may be improved is by supplying Cu as nanoparticles. The objective of our study was to establish the bioavailability of nanoparticle CuO compared with conventional CuO in dairy cows without or in combination with antagonists (S and Mo) in the diet. Fifty-six multiparous Holstein-Friesian cows that were 48-d (SD ± 17.4) post calving and yielding 40.6 (SD ± 6.9) kg milk/d were used in a 2 × 2 factorial design. The 4 treatment groups were N−; nano CuO; N+; nano CuO and antagonists; C−; conventional CuO and C+; conventional CuO and antagonists. The diets contained approximately 17 mg Cu/kg DM, diets with antagonists contained an additional 6 mg Mo/kg and 1 g S/kg DM compared with diets without added antagonists. Blood samples were taken at wk 0, 2, 4, 6, 10 and 16 and liver biopsy samples at wk 0 and 16. Data were analyzed as a 2 × 2 factorial design using wk 0 data as a covariate where appropriate using Genstat 18. There was no effect of dietary treatment on dry matter intake, live weight, body condition score or milk yield. Plasma Cu and Fe concentrations were not affected by dietary treatment whereas plasma Mo concentration was increased (P < 0.001) in cows receiving dietary antagonists. Dietary antagonists decreased liver Cu concentration in cows fed conventional CuO (C+) but in cows fed dietary antagonists and nano CuO (N+) liver Cu concentration was increased (P < 0.05). In cows fed diets without antagonists liver Cu concentration was increased when supplemented with either conventional CuO (C−) or nano CuO (N−). In conclusion, nano CuO has a greater bioavailability than conventional CuO in the presence of Cu antagonists, and supplying CuO as a nanoparticle had no effect on animal performance.