Background: Cisplatin-induced nephrotoxicity is an important problem of the cancer treatments. The major bioactive component of Nigella sativa, thymoquinone (TQ) might limit the nephrotoxic effect of cisplatin in low doses. However, it is not clear how it can affect the kidney as an anti-cytotoxic agent when administered in higher doses or in cisplatin co-treatment. Therefore, we examined the in vivo interactions between cisplatin and TQ by measuring serum cystatin C (cys C), creatinine and neutrophil gelatinase-associated lipocalin (NGAL) levels and analyzing the expression status of p53 and NGAL by immunohistochemistry. Methods: Wistar rats were divided into four groups: Control, TQ treatment (group II; 40 mg/kg i.p. for 5 days), cisplatin treatment (group III; 7 mg/kg, i.p. for at day 3) and TQ and cisplatin co-treatment (group IV). Results: Administration of 40 mg/kg TQ had no effect on serum kidney parameters. In cisplatin received group's serum creatinine level was insignificant, but serum Csy C and NGAL levels were significantly increased. All serum creatinine, NGAL and Cys C levels were increased in co-treatment of cisplatin and TQ. Additionally, in this group, renal tubular damage was found significantly higher than both control and only cisplatin-treated groups. The kidney immunohistochemistry staining of NGAL and p53 were significantly more intense in group IV rather than the others. Conclusions: This study showed that the administration of cisplatin and high dose of TQ act synergistically to produce nephrotoxicity and the involvement of apoptotic pathway and proximal tubule damage might be the leading cause of on this effect.