Our previous studies revealed that methane (CH ) induces adventitious rooting in cucumber. However, the corresponding molecular mechanism is still elusive. In this work, we discovered that CH triggered the accumulation of nitric oxide (NO) and thereafter cucumber adventitious rooting, mimicking the inducing effects of sodium nitroprusside (SNP) and NONOate (two NO-releasing compounds). Above mentioned responses were sensitive to NO scavenger(s), showing that the accumulation of NO and adventitious root development were respectively impaired. Inhibitor test and biochemical analysis suggested that endogenous NO mainly produced by mammalian NO synthase-like enzyme and diamine oxidases (DAO), might be required for adventitious root formation elicited by CH . Molecular evidence confirmed that CH -mediated induction of several marker genes responsible for adventitious root development, including CsDNAJ-1, CsCDPK1, CsCDPK5, cell division-related gene CsCDC6, and two auxin signaling genes, CsAux22D-like and CsAux22B-like, was casually dependent on NO signaling. The possible involvement of S-nitrosylation during the mentioned CH responses was preliminarily illustrated. Taken together, through pharmacological, anatomical and molecular approaches, it is suggested that NO might be involved in CH -induced cucumber adventitious rooting, and CH -eliciated NO-targeted proteins might be partially modulated at transcriptional and post-translational levels. Our work may increase the understanding of the mechanisms underlying CH -elicited root organogenesis in higher plants.