Cold storage/rewarming is an inevitable process for kidney transplantation from deceased donors, which correlates closely with renal ischemia–reperfusion injury (IRI) and the occurrence of delayed graft function. Histone deacetylases (HDAC) are important epigenetic regulators, but their involvement in cold storage/rewarming injury in kidney transplantation is unclear. In the present study, we showed a dynamic change of HDAC3 in a mouse model of kidney cold storage followed by transplantation. We then demonstrated that the selective HDAC3 inhibitor RGFP966 could reduce acute tubular injury and cell death after prolonged cold storage with transplantation. RGFP966 also improved renal function, kidney repair and tubular integrity when the transplanted kidney became the sole life-supporting graft in the recipient mouse. In vitro, cold storage of proximal tubular cells followed by rewarming induced remarkable cell death, which was suppressed by RGFP966 or knockdown of HDAC3 with shRNA. Inhibition of HDAC3 decreased the mitochondrial pathway of apoptosis and preserved mitochondrial membrane potential. Collectively, HDAC3 plays a pathogenic role in cold storage/rewarming injury in kidney transplantation, and its inhibition may be a therapeutic option.