Endothelial dysfunction contributes to the development of ungulate's laminitis. Although extensively studied in equines, the endothelial function is not fully examined in bovine digital veins (BDVs). BDVs were studied under isometric conditions to describe the acetylcholine (ACh) endothelium‐dependent relaxation. Concentration–response curves were constructed to phenylephrine, ACh, and sodium nitroprusside (SNP). Relaxation responses were evaluated using either phenylephrine or depolarizing high‐potassium Krebs solution (DKS) as precontraction agents. Endothelium denudation and incubation with L‐NAME (300 μM), indomethacin (10 μM) or both were used to explore endothelial‐mediated mechanisms. Endothelium denudation did not modify phenylephrine and SNP responses, however, significantly (p < 0.05) converted a relaxation (63.2 ± 5%) response to ACh into a contraction (30.3±9%). The ACh‐evoked relaxation was significantly (p < 0.05) reduced in the presence of indomethacin (37.5 ± 6%) and L‐NAME (6.40 ± 2%). The presence of both inhibitors abolished the ACh‐evoked relaxation. Although DKS caused a higher precontraction than phenylephrine, ACh‐evoked relaxation (22.4 ± 3.4%) was still observed and was reduced by the combination of inhibitors (7.0 ± 1.0%). The ACh endothelium‐dependent relaxation in BDVs is essentially mediated by nitric oxide and endothelium‐derived prostanoids. The BDV endothelium function is a dynamic component in the control of the bovine digital blood flow, particularly under endothelial dysfunction conditions when venoconstriction might lead to postcapillary resistance increase.