Background The kidneys are considered one of the most susceptible organs for adverse drug effects, particularly in post-transplant conditions. Tacrolimus (FK506), a calcineurin inhibitor immunosuppressant, is an essential component in the transplantation regimen. Despite that, nephrotoxicity is a severe drawback for its chronic utilization, where oxidative stress might be implicated. Kaempferol (KMF) is a natural flavonoid that has many adaptable biological activities, including antioxidant action. Objective Exploring the KMF protective effect on FK506-induced nephrotoxicity and the underlying role of calcineurin B1. Methods Twenty-four male albino-Wistar rats were randomly divided into three equal groups. The control group received solvents: propylene glycol, i.p. and 0.5% carboxymethyl cellulose, PO; FK506 group was injected with FK506 (0.6 mg/kg, i.p.), and FK506+KMF group was given FK506 (0.6 mg/kg, i.p.) and KMF (10 mg/kg, PO). The treatment regimen for all groups was once daily for 30 days. ELISA technique applied for measuring FK506 trough level and nephrotoxicity biomarkers in serum (cystatin C and urea) on days 15 and 30, and in kidney tissue homogenate (MDA and calcineurin B1) on day 30. Results In FK506-treated rats, the FK506 trough level was 7.84 ± 1.31 ug/l on day 15 and 9.54 ± 1.45 ug/l on day 30. FK506 use has significantly ( P <0.01) increased biomarkers levels of cystatin C (325% and 477%), urea (177% and 245%), MDA (1253%), except calcineurin B1 that has decreased (97%). The KMF combination has resulted in a significant reduction in the FK506 trough level by day 30 (6.79 ± 1.35 ug/l, P <0.01). KMF has significantly ameliorated the levels of cystatin C (46% and 73%, P <0.001), urea (38% and 68%, P< 0.001), MDA (75%, P <0.001), and calcineurin B1 (1833%, P <0.05). Conclusion Oxidative stress and calcineurin B1 are contributing factors in FK506-induced nephrotoxicity. Hence, inhibition of calcineurin enzyme is not limited to the immune cells. KMF could be a novel nephroprotective antioxidant.