A positional isomer of 3',5'-cAMP, 2',3'-cAMP, is produced by kidneys in response to energy depletion, and renal 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) metabolizes 2',3'-cAMP to 2'-AMP; 2',3'-cAMP is a potent opener of mitochondrial permeability transition pores (mPTPs), which can stimulate autophagy. Because autophagy protects against AKI, it is conceivable that inhibition of CNPase protects against ischemia-reperfusion (IR) –induced AKI. Therefore, we investigated renal outcomes, mitochondrial function, number, area, and autophagy in CNPase-knockout (CNPase^–/–) versus wild-type (WT) mice using a unique two–kidney, hanging–weight model of renal bilateral IR (20 minutes of ischemia followed by 48 hours of reperfusion). Analysis of urinary purines showed attenuated metabolism of 2',3'-cAMP to 2'-AMP in CNPase^–/– mice. Neither genotype nor IR affected BP, heart rate, urine volume, or albumin excretion. In WT mice, renal IR reduced ¹⁴C-inulin clearance (index of GFR) and increased renal vascular resistance (measured by transit time nanoprobes) and urinary excretion of kidney injury molecule-1 and neutrophil gelatinase–associated lipocalin. IR did not affect these parameters in CNPase^–/– mice. Histologic analysis revealed that IR induced severe damage in kidneys from WT mice, whereas histologic changes were minimal after IR in CNPase^–/– mice. Measurements of renal cardiolipin levels, citrate synthase activity, rotenone–sensitive NADH oxidase activity, and proximal tubular mitochondrial and autophagosome area and number (by transmission electron microscopy) indicted accelerated autophagy/mitophagy in injured CNPase^–/– mice. We conclude that CNPase deletion attenuates IR-induced AKI, in part by accelerating autophagy with targeted removal of damaged mitochondria.