Hydrogen sulfide (HS) is emerging as a potential messenger molecule involved in modulation of physiological processes in plants. Mitogen-activated protein kinase (MAPK) and nitric oxide (NO) are essential for abiotic stress signaling. This work investigated the effects of HS and the crosstalk between HS, MAPK and NO in cucumber roots under nitrate stress. The inhibitory effect of 140 mM nitrate on the growth of shoot and root was substantially alleviated by treatment with HS donor sodium hydrosulfide (NaHS), especially 100 μM NaHS. Treatment with 100 μM NaHS reduced malondialdehyde (MDA) and HO contents, ROS accumulation and increased the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX). CsNMAPK transcript level was up-regulated by NaHS treatment, while significantly decreased by propargylglycine (PAG, specific inhibitor of HS biosynthesis) and hypotaurine (HT, HS scavenger) in cucumber roots under nitrate stress. NO accumulation was increased by NaHS treatment under nitrate stress, but reduced by HT, PAG and PD98059, indicating that NO might function downstream of MAPK and HS. MAPK inhibitor PD98059 and NO scavenger (cPTIO) reversed the alleviating effect of HS by increasing MDA and HO contents, and decreasing antioxidant enzyme activities of SOD, CAT, POD, APX, and the endogenous HS contents and LCD activities under nitrate stress. In conclusion, HS played a protective role in cucumber seedlings under nitrate stress and MAPK/NO signaling were involved in the process by regulating antioxidant enzyme activities.