Tetraploid wheat (Triticum turgidum L. var. durum) cv. 'Langdon' (LDN) and its near-isogenic recombinant substitution line no. 68 (RSL no. 68) carrying the high grain protein gene Gpc-B1 from emmer wheat, were compared in three greenhouse experiments to establish in which way Gpc-B1 increases grain protein concentration (GPC). At anthesis, RSL no. 68 had higher soluble protein and amino acids concentrations in the flag leaf than LDN. At maturity, both lines presented a similar above ground biomass and grain yield. However, RSL no. 68 showed a higher total N content in ears, grain and chaff than LDN; N harvest index (NHI) was also higher because of a lower straw N concentration and higher grain N concentration. When both lines were grown with a low N supply, and when N supply was interrupted before anthesis, similar trends were observed but the differences in GPC were smaller. It is concluded that RSL no. 68 accumulates a higher GPC than LDN mainly because of a more efficient N remobilization from the leaves to the ears during grain filling.