Control of microvascular reactivity by 5-hydroxytryptamine (5-HT; serotonin) is complex and may depend on vascular bed type and 5-HT receptors. 5-HT receptors consist of seven families (5-HT1-5-HT7), with 5-HT2 predominantly mediating renal vasoconstriction. Regulation of cyclooxygenase (COX) and smooth muscle cell (SMC) intracellular Ca2+ concentration ([Ca2+]i) levels have been implicated in 5-HT-induced vascular reactivity. Although 5-HT receptor expression and circulating 5-HT levels are known to be dependent on postnatal age, control of neonatal renal microvascular function by 5-HT is unclear. The objective of this study is to use the translational swine model to examine the effects of 5-HT on newborn renal microcirculation. We demonstrate that 5-HT activates human transient receptor potential cation channel subfamily V member 4 (TRPV4) channels that were transiently expressed in Chinese hamster ovary cells. 5-HT2A is the predominant 5-HT2 receptor subtype in freshly isolated neonatal pig renal microvascular SMC. HC-067047 (HC), a selective TRPV4 blocker, attenuated cation currents induced by 5-HT in neonatal pig renal vascular SMC. HC also inhibited the 5-HT-induced increase in renal microvascular [Ca2+]i and constriction. Intrarenal artery infusion of 5-HT had minimal effects on systemic hemodynamics but reduced renal blood flow (RBF) and increased renal vascular resistance (RVR) in the pigs. Transcutaneous determination of glomerular filtration rate (GFR) indicated that kidney infusion of 5-HT reduced GFR. HC diminished 5-HT-induced alteration of RBF, RVR, and GFR. Moreover, plasma and urinary COX-1 and COX-2 levels in 5-HT-treated pigs were unchanged. These data suggest that activation of renal microvascular SMC TRPV4 channels by 5-HT impairs kidney function in neonatal pigs independently of COX production. NIH R01DK120595 and R01DK127625 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.