Coccidia of the genus Cystoisospora cause mild to severe diarrhoea in dogs. The effects of toltrazuril treatment on cystoisosporosis were studied under experimental and field conditions. Twenty-four puppies were experimentally infected each with 4 × 104 oocysts of the Cystoisospora ohioensis group. Three groups of six puppies were treated 3 dpi with 10, 20 or 30 mg/kg body weight of toltrazuril suspension (5%); the remaining six puppies served as non-treated controls. Toltrazuril suspension or microgranulate were given once in a dose of 10 or 20 mg/kg body weight, respectively, to naturally infected puppies in conventional dog breeding facilities, depending on the coproscopical evidence of infection. Oocyst excretion and clinical data were recorded. Under experimental conditions, the non-treated puppies excreted oocysts beginning at 6 dpi and suffered from catarrhalic to haemorrhagic diarrhoea. On 12 dpi, four of six non-treated puppies died. Irrespective of the dose, toltrazuril treatment totally suppressed oocyst excretion and no diarrhoea or other signs of disease were observed in the treated groups. Natural Cystoisospora infections were regularly found during the 3rd or 4th week of age in dog breeding facilities although not always associated with diarrhoea. A single oral application of toltrazuril abrogated oocyst shedding and the treated puppies remained generally coproscopically negative during the following 2–4 weeks. Cystoisospora is pathogenic for puppies and can induce severe disease. Natural infections are common in conventional dog breeding facilities. Toltrazuril treatment is suitable for controlling cystoisosporosis under experimental and field conditions. A single oral treatment for puppies in the 3rd or 4th week of age is recommended.