Soil humic substances are known to positively influence plant growth and nutrition. In particular, low-molecular fractions have been shown to increase NO3- uptake and PM H+-ATPase activity and alter expression of related genes. In this work, a water-extractable low-molecular humic fraction (WEHS) has been tested for its ability to affect molecular physiology of nitrate uptake in two maize inbred lines with different NUE. WEHS causes an acceleration of the increase in net nitrate uptake rate in both lines, almost halving the time needed to reach the maximal uptake capacity. Transcriptional analyses indicate that WEHS positively modulate some genes involved in nitrate uptake (NRT2.1 and MHA2) and assimilation (NR2). It appears that humic substances can favour nitrogen acquisition by improving uptake and assimilation of nitrate. Results point out the importance of considering the interactions between roots and soil components in order to get a better understanding of nutrient use by plants and to improve agricultural practices aiming at reducing input of fertilizers.