Humic substances (HS) have positive effects on plant physiology, but the molecular mechanisms underlying these events are only partially understood. HS exert auxin-like activity, but data supporting this hypothesis are under debate. To investigate the auxin-like activity of HS, we studied their biological effect on lateral root initiation in Arabidopsis thaliana. To this aim we characterised HS by means of DRIFT and 13C CP/MAS NMR spectroscopy, and measured their endogenous content of IAA. We then utilised a combination of genetic and molecular approaches to unravel HS auxin activity in the initiation of lateral roots. The data obtained using specific inhibitors of auxin transport or action showed that HS induce lateral root formation mostly through their ‘auxin activity’. These findings were further supported by the fact that HS used in this study activated the auxin synthetic reporter DR5::GUS and enhanced transcription of the early auxin responsive gene IAA19.