Iron deficiency (ID) defined as plasma ferritin <12 µg/L is associated with delayed cognitive development in early childhood and increased incidence of infections, however the longitudinal association between early life factors and ID in 18-month-old children in Denmark is unknown. This study aimed to determine the prevalence of ID and to describe risk factors associated with ID in healthy 18-month-old Danish children. Blood samples, anthropometric measurements and self-reported questionnaire data had been obtained in the birth cohort, Odense Child Cohort (OCC). The questionnaires were modified from those used in the DNBC, Danish National Birth Cohort. Plasma ferritin and C-reactive protein in venous, non-fasting samples were analysed in the final sample size of 370 children after exclusion of 79 children due to chronic disease, acute infection, C-reactive protein >10 mg/L, twin birth or prematurity. Associations with ID were analysed by logistic regression, adjusting for sex, maternal education, duration of partial breastfeeding and current intake of milk, fish and meat. Overall, 56 children had ID (15.1%). Factors associated with increased risk were exclusive breastfeeding beyond 4 months (OR 5.97, 95%CI [1.63; 21.86]) and no intake of oral iron supplements from 6-12 months (OR 3.99, 95%CI [1.33; 11.97]. Duration of partial breastfeeding and current diet were not associated with ID. In conclusion, the ID prevalence was 15.1%, and both exclusive breastfeeding beyond 4 months and no intake of oral iron supplements from 6-12 months were associated with increased risk for ID in 18-month-old children.