Spinal afferent neurons, with endings in the intestinal mesenteries, have been shown to respond to changes in vascular perfusion rates. The mechanisms underlying this sensitivity were investigated in an in vitro preparation of the mesenteric fan devoid of connections with the gut wall. Afferent discharge increased when vascular perfusion was stopped (“flow off”), a response localized to the terminal vessels just prior to where they entered the gut wall. The flow-off response was compared following pharmacological manipulations designed to determine direct mechanical activation from indirect mechanisms via the vascular endothelium or muscle. Under Ca²⁺-free conditions, responses to flow off were significantly augmented. In contrast, the myosin light chain kinase inhibitor wortmannin (1 μM, 20 min) did not affect the flow-off response despite blocking the vasoconstriction evoked by 10 μM l-phenylephrine. This ruled out active tension, generated by vascular smooth muscle, in the response to flow off. Passive changes caused by vessel collapse during flow off were speculated to affect sensory nerve terminals directly. The flow-off response was not affected by the N-, P-, and Q-type Ca²⁺ channel blocker ω-conotoxin MVIIC (1 μM intra-arterially) or the P2X receptor/ion channel blocker PPADS (50 μM). However, ruthenium red (50 μM), a blocker of nonselective cation channels, greatly reduced the flow-off response and also abolished the vasodilator response to capsaicin. Our data support the concept that mesenteric afferents sense changes in vascular flow during flow off through direct mechanisms, possibly involving nonselective cation channels. Passive distortion in the fan, caused by changes in blood flow, may represent a natural stimulus for these afferents in vivo.