In summary, we have elucidated the molecular mechanism of "template-dependent inhibition" exerted by Remdesivir on the NTP incorporation in SARS-CoV-2 RdRp by comprehensive analysis based on extensive MD simulations. Our computational results suggested that the Remdesivir at the template strand did not directly abolish the complementary NTP incorporation at the active site. Instead, translocation of Remdesivir from +2 to +1 site was inhibited, and thus the NTP incorporation became inefficient. Specifically, the dynamic translocation of Remdesivir from +2 site to +1 site is kinetically hindered due to the clash between the 1'-cyano group of Remdesivir and the side chain of V557, while the V557L mutation tends to reduce the inhibitory effect by rotating its side chain away from the translocation pathway. This finding not only matches with the experimental observation that SARS-CoV-2 RdRp upon V557L mutation would gain drug resistance to Remdesivir, but also provides the underlying mechanism at the unprecedented molecular level. As the template-dependent inhibition has been observed across diverse viral RNA polymerases , our finding may shed light on the complex inhibition mechanism in other RdRps. Overall, our studies have provided valuable molecular insights into the inhibitory mechanism when Remdesivir is embedded in the template strand and underlined the role of 1'-cyano substitution in exerting the inhibitory effect. This offers an alternative strategy for the rational design of antiviral drugs targeted at SARS-CoV-2 RdRp during the synthesis of the second RNA strand for viral transcription and genome synthesis.