Observational studies frequently report phenotypic associations between low resting heart rate (RHR) and higher levels of antisocial behaviour (ASB), although it remains unclear whether this relationship reflects causality. To triangulate evidence, we conducted two-sample univariable Mendelian randomisation (MR), multivariable MR and linkage disequilibrium score regression (LDSC) analyses. Genetic data were accessed from published genome-wide association studies (GWAS) for RHR (n = 458,835) and ASB (n = 85,359) for the univariable analyses, along with a third GWAS for heart rate variability (HRV; n = 53,174) for all other analyses. Genome-wide significant (p< 5 × 10−8) single-nucleotide polymorphisms associated with RHR (n = 278) were selected as instrumental variables and the outcome was a composite measure of ASB. No causal association was observed between RHR and ASB (BIVW=  − 0.0004,p= 0.841). The multivariable MR analyses including RHR and HRV also suggested no causal associations (BIVW= 0.016,p= 0.914) and no genetic correlations between the heart rate measures and ASB were observed using LDSC (rg= 0.057,p= 0.169). Sensitivity analyses suggested that our results are not likely to be affected by heterogeneity, pleiotropic effects, or reverse causation. These findings suggest that individual differences in autonomic nervous system functioning indexed by RHR are not likely to directly contribute to the development of ASB. Therefore, previously observed associations between RHR and ASB may arise from confounding, reverse causation, and/or additional study characteristics. Further causally informative longitudinal research is required to confirm our findings, and caution should be applied when using measures of RHR in interventions targeting ASB.