An enterotoxigenic Escherichia coli (ETEC) vaccine designed to prevent diarrhoea was inoculated into dairy cows, and the occurrence of clinical mastitis was investigated for 2 years. Half of 480 cows in five farms were subcutaneously inoculated with ETEC vaccine (Imocolibov) twice with a 1-month interval in 2007 and 2008. Fisher's exact test and survival (time to event) analysis with the log-rank test were used to compare vaccinates and controls. In 2007, there was no significant difference in the incidence rate of mastitis between vaccinate (20·3%) and control (17·1%) cows. The rate of death or culling due to mastitis was lower in vaccinated cows (7·4%) than in control cows (29·2%, P=0·07, Fisher's exact test; P=0·02, log-rank test). In 2008, there was no significant difference in both the incidence rate of mastitis and the rate of death or culling due to mastitis. Milk productivity was compared between vaccinates and controls in one farm. Multi-way analysis of variance (ANOVA) was performed for the amount of 4% fat-corrected milk, and there was no significant difference between vaccinates and controls. These results suggest that ETEC vaccine inoculation reduces death or culling due to mastitis, whereas no preventive effect on the development of mastitis was observed.