Recent studies have reported an increased risk of autism among second-born children conceived <12 versus >36 months after the birth of a sibling. Confirmation of this finding would point to inter-pregnancy interval (IPI) as a potentially modifiable risk factor for autism. This study evaluated the relationship between IPI and autism spectrum disorder (ASD) risk in a Wisconsin birth cohort of 31,467 second-born children, of whom 160 resided in the study area and were found to have ASD at age 8 years. In adjusted analyses, both short (<12) and long (>84 month) IPIs were associated with a two-fold risk of ASD relative to IPIs of 24-47 months (p < 0.05). The long IPI association was partially confounded by history of previous pregnancy loss.