Utilization of organic nitrogen (N) is an important aspect of plant N assimilation and has potential application in sustainable agriculture. The aim of this study was to investigate the plant growth, C and N accumulation in leaves and roots of tomato seedlings in response to inorganic (NH4+-N, NO3--N) and organic nitrogen (Gly-N). Different forms of nitrogen (NH4+-N, NO3--N, Gly-N) were supplied to two tomato cultivars (Shenfen 918 and Huying 932) using a hydroponics system. The plant dry biomass, chlorophyll content, root activity, total carbon and nitrogen content in roots and leaves, and total N absorption, etc. were assayed during the cultivation. Our results showed that no significant differences in plant height, dry biomass, and total N content were found within the first 16 d among three treatments; however, significant differences in treatments on 24 d and 32 d were observed, and the order was NO3--N > GIy-N > NH4+-N. Significant differences were also observed between the two tomato cultivars. Chlorophyll contents in the two cultivars were significantly increased by the GIy-N treatment, and root activity showed a significant decrease in NH4+-N treatment. Tomato leaf total carbon content was slightly affected by different N forms; however, total carbon in root and total nitrogen in root and leaf were promoted significantly by inorganic and organic N. Among the applied N forms, the increasing effects of the NH4+-N treatment were larger than that of the Gly-N. In a word, different N resources resulted in different physiological effects in tomatoes. Organic nitrogen (e.g., Gly-N) can be a proper resource of plant N nutrition. Tomatoes of different genotypes had different responses under organic nitrogen (e.g., Gly-N) supplies.