Alkamides are small bioactive lipid signals with a wide distribution in plants. In this report, the role of nitric oxide (NO) in the alterations induced by N-isobutyl decanamide on the Arabidopsis (Arabidopsis thaliana) root system architecture (RSA) was investigated. We first compared the effects of N-isobutyl decanamide and NO donors sodium nitropruside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) on root morphogenetic processes. Both N-isobutyl decanamide and NO donors modulated RSA in a similar way and in a dose-dependent manner, inhibiting primary root growth and promoting lateral root primordia (LRP) formation. RSA alterations induced by N-isobutyl decanamide correlated with NO accumulation in the primary root tip and in developing lateral roots. Morphogenetic effects of N-isobutyl decanamide decreased when NO scavengers were supplied to alkamide-treated seedlings. N-Isobutyl decanamide-regulated root architectural changes were also investigated in mutants defective in NO biosynthesis, nia1 nia2, and NO signalling, Atnoa1, and in the alkamide-resistant mutant drr1. The nia1 nia2 and Atnoa1 mutants were indistinguishable in primary root growth inhibition by the alkamide when compared with wild-type (WT) seedlings, but showed reduced lateral root responses. The drr1 mutant was less sensitive in both primary root growth inhibition and LRP induction by NO donors than WT seedlings. Detailed DR5:uidA and BA3:uidA marker analysis showed that N-isobutyl decanamide and its interacting signals jasmonic acid and NO act downstream or independently of auxin-responsive gene expression to promote LRP formation. Our results provide compelling evidence that NO is an intermediate in alkamide signaling mediating RSA adjustment in Arabidopsis.