Aims Increasing evidence shows that root system growth is strongly affected by different nitrogen (N) signals and the available N source in the rhizosphere. Molybdenum (Mo), an essential trace element, plays a key role in N assimilation and metabolism. However, Mo efficacy may vary with different N sources. The present experiment was performed to examine the Mo role on root system growth in winter wheat under different N sources. Methods A hydroponic experiment was conducted consisting of two winter wheat cultivars; Mo-efficient (97003) and Mo-inefficient (97014) under two Mo levels (0 and 1 μM) and three N sources (NO 3 − , NH 4 NO 3 or NH 4 + ). Results The results showed that Mo supply increased root dry weight and other morphological traits, nitrate reductase (NR) activities, nitric oxide (NO) contents, total N concentration and the expressions of nitrate transporter ( NRT ) genes under various N sources, however, such effects were in the order of NH4NO3 > NO 3 −  > NH 4 + , suggesting that Mo fertilizer shows more complementary effects towards NO 3 − form of N than sole NH 4 + in winter wheat. Conclusions These findings imply that Mo plays a significant role in increasing the root system growth of wheat through NO production, efficient N uptake and assimilation, and regulation of NRT gene expressions, especially under NH4NO3 nutrition.