While fertigation can increase fertilizer use efficiency, there is an uncertainly as to whether the fertilizer should be introduced at the beginning of the irrigation or at the end, or introduced during irrigation. Our objective was to determine the effect of different fertigation schemes on nitrogen (N) uptake and N use efficiency (NUE) in cotton plants. A pot experiment was conducted under greenhouse conditions in year 2004 and 2005. According to the application timing of nitrogen (N) fertilizer solution and water (W) involved in an irrigation cycle, four nitrogen fertigation schemes [nitrogen applied at the beginning of the irrigation cycle (N-W), nitrogen applied at the end of the irrigation cycle (W-N), nitrogen applied in the middle of the irrigation cycle (W-N-W) and nitrogen applied throughout the irrigation cycle (N&W)] were employed in a completely randomized design with four replications. Cotton was grown in plastic containers with a volume of 84 l, which were filled with a clay loam soil and fertilized with 6.4 g of N per pot as unlabeled and 15N-labeled urea for 2004 and 2005, respectively. Plant total dry matter (DM) and N content in N-W was significantly higher than in N&W in both seasons, but these were not consistent for W-N and W-N- W treatments. In year 2005, a significantly higher nitrogen derived from fertilizer (NDFF) for the whole plant was found in W-N and N-W than that in W-N-W and N&W. Fertigation scheme had a consistent effect on total NUE: N-W had the highest NUE for the whole plant, but this was not significantly different from W-N. Treatments W-N and W-N-W had similar total NUE, and N&W had the lowest total NUE. After harvesting, the total residual fertilizer N in the soil was highest in W-N, lowest in N-W, but this was not significantly different from N&W and W-N-W treatments. Total residual NO3-N in the soil in N&W and W-N treatments was 20.7 and 21.2% higher than that in N-W, respectively. The total 15 N recovery was not statistically significant between the four fertigation schemes. In this study, the fertigation scheme N-W (nitrogen applied at the beginning of an irrigation cycle) increased DM accumulation, N uptake and NUE of cotton. This study indicates that Nitrogen application at the beginning of an irrigation cycle has an advantage on N uptake and NUE of cotton. Therefore, NUE could be enhanced by optimizing fertilization schemes with drip irrigation.