Background: Renal ischemia-reperfusion (I/R) injury is a major cause of acute renal failure (ARF). The transcription factor nuclear factor-κB (NF-κB) has been implicated as a key mediator of reperfusion injury. Activation of NF-κB is dependent upon the phosphorylation of its inhibitor, IκB, by the specific inhibitory κB kinase (IKK) subunit, IKKβ. We hypothesized that ischemic preconditioning (IPC) reduces acute renal damage following I/R injury by inhibiting activation of IKKβ. As neutrophil gelatinase-associated lipocalin (NGAL), an early predictive biomarker of acute kidney injury, is regulated by NF-κB, we approached the relationship between NGAL and IKKβ. Method: Thirty male Sprague-Dawley rats were randomly divided into 3 groups after right kidney nephrectomy. Group A rats were sham-operated controls. Group B rats were 45-min ischemic in the left renal artery while Group C rats were pre-treated with 3 cycles of 2-min ischemia and 5-min reperfusion. All the rats were sacrificed at 24 h after reperfusion. We harvested kidneys and serum to do further analysis, including histological and functional parameters, expressions of NGAL and IKKβ in renal tissues. Results: Compared with rats subjected to I/R injury, pre-treated rats had a significant decrease in serum creatinine level (Scr) and tubulointerstitial injury scores (Scr, 86.79 ± 12.98 vs. 205.89 ± 19.16 μmol/l, p < 0.01; tubulointerstitial injury scores, 1.3 ± 0.48 vs. 3.8 ± 0.79, p < 0.01). In addition, expressions of IKKβ (0.95 ± 0.21 vs. 1.74 ± 0.17, p < 0.05) and NGAL (1.71 ± 0.032 vs. 2.66 ± 0.078, p < 0.05) at renal tubule in pre-treated rats were attenuated significantly compared with rats subjected to ischemia-reperfusion injury. Moreover, our study showed that IKKβ and NGAL were in positive correlation (R = 0.965 > R0.01(30) = 0.448, p < 0.01). Conclusions: The evidence suggests that IKKβ may play a role in renal I/R injury and give rise to the generation of NGAL. It appears that IPC may attenuate renal injury and the expression of NGAL following acute I/R injury. IKKβ may offer a clinically accessible target for preventing renal injury following I/R.