--- - |2+ Abstract Aims Klotho interacts with various membrane proteins, such as receptors for transforming growth factor (TGF)‐β and insulin‐like growth factor (IGF), to alter their function. Renal expression of klotho is diminished in diabetes. The present study examined whether exogenous klotho protein supplementation ameliorates kidney injury and renin–angiotensin system (RAS) in db/db mice. Methods We investigated the effects of klotho supplementation on diabetic kidney injury and RAS. Recombinant human klotho protein (10 μg/kg/d) was administered to db/db mice daily. Results Klotho protein supplementation reduced kidney weight, systolic blood pressure (SBP), albuminuria, glomerular filtration rate, and 8‐epi‐prostaglandin F2α excretion without affecting body weight. Although klotho supplementation did not alter glycated albumin, it reduced renal angiotensin II levels associated with reduced renal expression of angiotensinogen. Klotho supplementation improved renal expression of superoxide dismutase (SOD), and endogenous renal expression of klotho. Klotho supplementation reduced the levels of hypoxia‐inducible factor, phosphorylated Akt, and phosphorylated mTOR and decreased the renal expression of TGF‐β, tumour necrosis factor (TNF), and fibronectin. Conclusions These data indicate that klotho supplementation reduces blood pressure and albuminuria along with ameliorating renal RAS activation in db/db mice. Furthermore, these results suggest that klotho inhibits IGF signalling, induces SOD expression to reduce oxidative stress, and suppresses Akt‐mTOR signalling to inhibit abnormal kidney growth. Collectively, the results suggest that klotho inhibits TGF‐β and TNF signalling, resulting in a decline in renal fibrosis. - 'Acta Physiologica, EarlyView. '