The dramatic land use and cover change (LUCC) is one of the major threats to biological conservation as landscape connectivity decreases and necessary ecological process is broken in rapidly urbanized regions. Although most ecological conservation plans aim to restore connectivity based on modeled data or empirical field investigation, few evaluate the changes of landscape connectivity quantitatively, especially from the perspective of regional LUCC analysis and simulation of potential ecological process. We took rapidly urbanized Nanjing in eastern China as the case, simulated potential ecological processes and mapped landscape corridors with minimum cumulative resistance (MCR) model. We then calculated the cumulative resistance values (CRVs) of the indicated corridors to reflect the quantitative changes of landscape connectivity. The potential least-cost paths (LCPs) of MCR model indicated the directions of the landscape corridors and they mostly followed the continuous rivers. Although most LCPs showed similar directions in 2000 and 2015, some significantly changed, especially those to the suburban targets where dramatic LUCCs formed barriers and caused obvious landscape fragmentation. The quantitative results of CRVs on the 2000’s LCPs showed a great increase from 2000 to 2015, indicating the decrease of landscape connectivity related with significant LUCCs from non-construction to construction lands. However, the CRVs of the 2015’s LCPs were mostly lower and did not change too much in 2000 and 2015. From the perspective of LUCCs, most inner buffers of the LCPs were dominated by waters and showed fewer changes comparing with outer buffers, especially those of the 2015’s LCPs. Therefore, it is concluded the 2015’s LCPs can maintain higher and more stable landscape connectivity as more ecologically suitable lands exist there in 2000 and 2015. To be specific, it is of lower cost to design the landscape corridors at the 0–50 m buffers, where limited land use adjustments are required in some new towns or industrial parks.