By using pharmacological and molecular approaches, we discovered the involvement of HO-1 in NaHS-induced lateral root formation in tomato seedlings. Heme oxygenase-1 (HO-1) and hydrogen sulfide (H2S) regulate various responses to abiotic stress and root development, but their involvement in the simultaneous regulation of plant lateral root (LR) formation is poorly understood. In this report, we observed that the exogenously applied H2S donor sodium hydrosulfide (NaHS) and the HO-1 inducer hemin induce LR formation in tomato seedlings by triggering intracellular signaling events involving the induction of tomato HO-1 (SlHO-1), and the modulation of cell cycle regulatory genes, including the up-regulation of SlCDKA;1 and SlCYCA2;1, and simultaneous down-regulation of SlKRP2. The response of NaHS in the induction of LR formation was impaired by the potent inhibition of HO-1, which was further blocked when 50 % saturation of carbon monoxide (CO) aqueous solution, one of the catalytic by-products of HO-1, was added. Further molecular evidence revealed that the NaHS-modulated gene expression of cell cycle regulatory genes was sensitive to the inhibition of HO-1 and reversed by cotreatment with CO. The impairment of LR density and length as well as lateral root primordia number, the decreased tomato HO-1 gene expression and HO activity caused by an H2S scavenger hypotaurine were partially rescued by the addition of NaHS, hemin and CO (in particular). Together, these results revealed that at least in our experimental conditions, HO-1 might be involved in NaHS-induced tomato LR formation. Additionally, the use of NaHS and hemin compounds in crop root organogenesis should be explored.