Holoprosencephaly (HPE) is a congenital defect of the brain characterized by incomplete cleavage of the embryonic forebrain into left and right hemispheres. Although a substantial proportion of cases of HPE can be attributed to genetic abnormalities, the etiology in many cases remains unknown, with non-genetic risk factors believed to be important contributors. Due to the low birth prevalence of this defect, it has proven difficult to conduct studies of sufficient size to identify risk factors with certainty. This article provides a summary of non-genetic risk factors for HPE that have been investigated in case reports and case series, animal studies, and epidemiologic studies, including maternal illnesses, therapeutic and non-therapeutic exposures, nutritional factors, and sociodemographic factors. The article also highlights challenges in study design and further areas for research to better understand the etiology of HPE.