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Objective To investigate the optimal timing of acute kidney injury environment for proliferation and differentiation of mesenchymal stem cells (MSCs).Methods MSCs were co-cultured with kidney homogenate at different ischemia/reperfusion (I/R) time points.The proliferation was reflected by CCK-8 assay and cell number counting.The differentiation was reflected by cell shape and CK-18 RNA expression.Results After 15d culture,shape of MSCs cultured with kidney homogenate at 72h time point was much more round and ellipsoidal,and the cells had a cobble like appearance.Cell number,CCK-8 and CK-18 level of MSCs cultured with kidney homogenate at 72h time point was significantly higher comparing with other time points.Conclusions 72h post I/R was the optimal timing for proliferation and differentiation of MSCs.