Background: The extraction and processing of copper and cobalt in the African Copperbelt in the Democratic Republic of Congo have led to substantial environmental pollution, causing concerns about possible adverse effects on human health, including birth defects. Cases: We report three neonates with clinically diagnosed holoprosencephaly who were part of a case-control study performed in Lubumbashi between February 2013 and February 2015. One mother had a high concentration of uranium in urine, and high manganese concentrations were found in blood of another mother and in cord blood of one infant. Two of the three fathers had a mining-related job. Discussion: We hypothesize that these cases of holoprosencephaly were connected to mining-related pollution, possibly via epigenetic alterations induced by paternal occupational exposure to toxic metals. Katanga, metals, mining, uranium