Background: Second-degree atrioventricular (AV) block at rest is very common in horses. The underlying molecular mechanisms are unexplored, but commonly attributed to high vagal tone. Aim: To assess whether AV block in horses is due to altered expression of the effectors of vagal signalling in the AV node, with specific emphasis on the muscarinic acetylcholine receptor (M₂) and the G protein-gated inwardly rectifying K⁺ (GIRK4) channel that mediates the cardiac I_K,ACh current. Method: Eighteen horses with a low burden of second-degree AV block (median 8 block per 20 h, IQR: 32 per 20 h) were assigned to the control group, while 17 horses with a high burden of second-degree AV block (median: 408 block per 20 h, IQR: 1,436 per 20 h) were assigned to the AV block group. Radiotelemetry ECG recordings were performed to assess PR interval and incidence of second-degree AV block episodes at baseline and on pharmacological blockade of the autonomic nervous system (ANS). Wenckebach cycle length was measured by intracardiac pacing (n = 16). Furthermore, the expression levels of the M₂ receptor and the GIRK4 subunit of the I_KACh channel were quantified in biopsies from the right atrium, the AV node and right ventricle using immunohistochemistry and machine learning-based automated segmentation analysis (n = 9 + 9). Results: The AV block group had a significantly longer PR interval (mean ± SD, 0.40 ± 0.05 s; p < 0.001) and a longer Wenckebach cycle length (mean ± SD, 995 ± 86 ms; p = 0.007) at baseline. After blocking the ANS, all second-degree AV block episodes were abolished, and the difference in PR interval disappered (p = 0.80). The AV block group had significantly higher expression of the M₂ receptor (p = 0.02), but not the GIRK4 (p = 0.25) in the AV node compared to the control group. Both M₂ and GIRK4 were highly expressed in the AV node and less expressed in the atria and the ventricles. Conclusion: Here, we demonstrate the involvement of the m₂R-I_K,ACh pathway in underlying second-degree AV block in horses. The high expression level of the M₂ receptor may be responsible for the high burden of second-degree AV blocks seen in some horses.