Key points Arginine vasopressin (AVP) stimulates the release of enteroendocrine L‐cell derived hormones glucagon‐like peptide‐1 (GLP‐1) and peptide YY (PYY) in vitro from mouse and human colons. This is mediated by the AVP receptor 1B, which is highly enriched in colonic L‐cells and linked to the elevation of L‐cell calcium and cAMP concentrations. By means of Ussing chambers, we show that AVP reduced colonic anion secretion, although this was blocked by a specific neuropeptide Y receptor Y1 receptor antagonist, suggesting that L‐cell‐released PYY acts locally on the epithelium to modulate fluid balance. In human serum samples, PYY concentrations were higher in samples with raised osmolality and copeptin (surrogate marker for AVP). These findings describe, for the first time, the role of L‐cells in AVP regulated intestinal fluid secretion, potentially linking together hormonal control of blood volume and blood glucose levels, and thus adding to our understanding of the complex pathways involved in the gut hormonal response to different stimuli. Abstract Arginine vasopressin (AVP) regulates fluid balance and blood pressure via AVP receptor (AVPR)2 in the kidney and AVP receptor 1A in vascular smooth muscle. Its role in intestinal function has received less attention. We hypothesized that enteroendocrine L‐cells producing glucagon‐like peptide 1 (GLP‐1) and peptide YY (PYY) may be a target of AVP and contribute to the control of fluid balance. Avpr1b expression was assessed by quantitative RT‐PCR on flourescence‐activated cell sorting‐isolated L‐ and control cells and was enriched in colonic L‐cells. AVP stimulated GLP‐1 and PYY release from primary cultured murine and human colonic cells and was associated with elevated calcium and cAMP concentrations in L‐cells as measured in cultures from GLU‐Cre/ROSA26‐GCaMP3 and GLU‐Epac2camps mice. An antagonist of AVPR1B reduced AVP‐triggered hormone secretion from murine and human cells. In Ussing chambers, basolaterally applied AVP reduced colonic anion secretion and this effect was blocked by a specific neuropeptide Y receptor Y1 (NPY1R) antagonist. In human serum, PYY concentrations were higher in samples with raised osmolality or copeptin (a surrogate marker for AVP). In conclusion, we propose that AVP activates L‐cell AVPR1B, causing GLP‐1 and PYY secretion. PYY in turn reduces colonic anion secretion via epithelial NPY1R. Our data suggest L‐cells are active players in the hypothalamic control of intestinal fluid homeostasis, providing a potential link between the regulation of blood volume/pressure/osmolality and blood glucose.