Grain protein has recently received attention in several countries because of a premium associated to increased protein concentration (PC) in grains. Nitrogen (N) fertilizer management with its timing of application and rates plays a crucial role in increasing PC. The objectives of the present study were to: (1) quantify the effects of different N fertilizer rates and timing of application on grain yield and grain protein of six durum wheat cultivars, and (2) identify the best cultivar in terms of net return, yield, and protein content in typical pedo-climatic condition of southern Italy. The field experiments were conducted in Foggia during three growing seasons (2002–2003, 2003–2004, and 2004–2005). Results from this study showed that N application at sowing did not influence the cultivars performance. N applied at stem elongation increased yield and protein content provided there is adequate soil water for N uptake. In absolute terms, the best combination was the cultivar Svevo managed using 104 kg N ha split between tillering and stem-elongation stage. The excessive N splitting reduces yield and does not provide any improvement in terms of protein content.