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Simple Summary: Transcutaneous auricular vagal nerve stimulation is a non-invasive technique used in human medicine to prevent or treat post-operative ileus. The vagus nerve plays a crucial role in increasing intestinal motility and reducing the inflammatory response in sepsis models; thus, its stimulation constitutes an attractive strategy for modulating intestinal motility and immune response after abdominal surgery in both humans and horses. The technique has never been described in the equine species before. This study describes transcutaneous auricular vagal nerve stimulation in healthy non-sedated horses, assessing its feasibility and effect on heart rate variability (HRV). HRV was analysed as an indirect method to identify parasympathetic stimulation of the treatment. Auricular vagal nerve stimulation is an economical procedure that is easy to perform. However, it was poorly tolerated in awake horses. In the mares that tolerated the procedure, its effect on the autonomic system confirmed the potential stimulation of vagal activity, which can be of interest in preventing post-operative ileus in horses with inflammation or after exploratory laparotomy. This study aimed to evaluate the feasibility of transcutaneous auricular vagal nerve stimulation (tAVNS) in healthy horses and its effect on heart rate variability (HRV). The study comprised three phases: the selection of mares, their acclimatization to the tAVNS, and the stimulation phase. Stimulation was performed with two electrodes positioned on the right pinna. The settings were 0.5 mA, 250 μs, and 25 Hz for pulse amplitude, pulse width, and pulse frequency, respectively. HRV was analysed before (B1), during (T), and after (B2) the tAVNS. From the 44 mares initially included, only 7 completed the three phases. In these mares, the heart rate (HR) was significantly lower, and frequency domain parameters showed an increased parasympathetic tone in B2 compared with B1. However, in 3/7 mares, the HR was significantly higher during T compared with B1 and B2, compatible with a decreased parasympathetic tone, while in 4/7 mares, the HR was significantly lower and the parasympathetic nervous system index was significantly higher during T and B2 compared with B1. The tAVNS is an economical and easy procedure to perform and has the potential to stimulate vagal activity; however, it was poorly tolerated in the mares included in this study. [ABSTRACT FROM AUTHOR]