Lowered temperatures may reduce the root water uptake of tropical trees at high elevations through several mechanisms; however, field studies to test their relevance are lacking. We measured sap flux density ( J) in small-diameter tree roots across a 2000-m elevation transect in a tropical mountain forest for quantifying the effects of temperature ( T a ), VPD and soil moisture ( θ) on root water flow and uptake at different elevations. Recently developed miniature heat balance-sap flow gauges were applied to roots of about 10 mm in diameter in mountain forest stands at 1050, 1890 and 3060 m a.s.l. in the Ecuadorian Andes and the measured flow was related to anatomical properties of the root xylem. Between 1050 and 3060 m, mean J decreased to about a third. VPD was the most influential environmental factor controlling J at 1050 and 1890 m, while T a was the key determinant at 3060 m. Large vessels were absent in the root xylem of high-elevation trees which resulted in a 10-fold decrease of theoretical hydraulic conductivity ( k h theor) between 1050 and 3060 m. We conclude that both physical limitations (reduced VPD, increased viscosity of water) and biological constraints (large decrease of k h theor) result in a significantly reduced J and root water uptake of the trees in high-elevation tropical forests.