Focal adhesion dynamics and protrusion of the leading edge are critical for cancer cell migration and wound healing of epithelial cells. Massive actin polymerization at the leading edge generates a forward protrusion force at the cell membrane. At the same time, the entire actin network moves toward the cell center, which is called the retrograde actin flow. The interaction between the actin flows and the focal adhesions (FAs) has been proposed to enhance the membrane protrusion. However, how FAs influence local retrograde flow is not fully understood. In this study, we developed a new fluorescence single-molecule speckle (SiMS) microscopy method for actin, which enables in vivo nanometer-scale displacement analysis with a low localization error of ±8-8.5 nm (Mol. Biol. Cell, 2014). Using the new method, we revealed for the first time that short-lived lamellipodial actin filaments (lifetime < 10 s), which move over a few hundred nanometers, flow with speeds similar to the other actin filaments. These results suggest that actin filaments in lamellipodia form a tightly connected network experiencing a uniform flow rate, regardless of individual filament lifetimes. On the other hand, mature FAs, but not nascent adhesions, locally obstruct the retrograde flow. Interestingly, the actin flow in front of mature FAs is fast and biased toward FAs, suggesting that mature FAs attract the flow in front. The results of this study provide a novel concept on the relationship between focal adhesions and the local actin flows: The interaction of mature FAs with the actin network might be more complex than a passive stick-slip interaction as previously suggested. Instead, mature FAs may be actively engaged in pulling and remodeling the local actin network.