Objective The G‐allele of the ‐1019C/G (rs6295) promoter polymorphism of the serotonin receptor 1A (HTR1A) gene has been implicated in anxiety; however, the underlying neurophysiological processes are still not fully understood. Recent evidence indicates that low parasympathetic (vagal) tone is predictive of anxiety. We thus conducted a structural equation model (SEM) to examine whether the HTR1A rs6295 variant can affect anxiety by altering parasympathetic nervous activity. Method A sample of 1141 drug‐free healthy Han Chinese was recruited for HTR1A genotyping. Autonomic nervous function was assessed by short‐term spectral analysis of heart rate variability (HRV). Anxiety and stress levels were evaluated by the Beck Anxiety Inventory (BAI) and the Perceived Stress Scale (PSS) respectively. Results The number of the HTR1A G allele was inversely correlated with high‐frequency power (HF), a parasympathetic index of HRV. The HF index was negatively associated with BAI scores. Furthermore, the good‐fitting SEM, adjusting for confounding variables (e.g., age and PSS levels), revealed a significant pathway linking rs6295 variant to BAI scores via HF index modulation. Conclusion These results are the first to show that HTR1A ‐1019C/G polymorphism influences anxiety levels by modulating parasympathetic tone, providing a neurophysiological insight into the role of HTR1A in human anxiety.