Adenosine diphosphate ribosylation (ADP-ribosylation; ADPr), the addition of ADP-ribose moieties onto proteins and nucleic acids, is a highly conserved modification involved in a wide range of cellular functions, from viral defence, DNA damage response (DDR), metabolism, carcinogenesis and neurobiology. Here we study MACROD1 and MACROD2 (mono-ADP-ribosylhydrolases 1 and 2), two of the least well-understood ADPr-mono-hydrolases. MACROD1 has been reported to be largely localized to the mitochondria, while the genomic locus has been associated with various neurological conditions such as autism, attention deficit hyperactivity disorder (ADHD) and schizophrenia; yet the potential significance of disrupting these proteins in the context of mammalian behaviour is unknown. Therefore, here we analysed both and gene knockout (KO) mouse models in a battery of well-defined, spontaneous behavioural testing paradigms. Loss of resulted in a female-specific motor-coordination defect, whereas disruption was associated with hyperactivity that became more pronounced with age, in combination with a bradykinesia-like gait. These data reveal new insights into the importance of ADPr-mono-hydrolases in aspects of behaviour associated with both mitochondrial and neuropsychiatric disorders.