The actin filament network immediately under the plasma membrane at the leading edge of rapidly moving cells consists of short, branched filaments, while those deeper in the cortex are much longer and are rarely branched. Nucleation by the Arp2/3 complex activated by membrane-bound factors (Rho-family GTPases and PIP(2)) is postulated to account for the formation of the branched network. Tropomyosin (TM) binds along the sides of filaments and protects them from severing proteins and pointed-end depolymerization in vitro. Here, we show that TM inhibits actin filament branching and nucleation by the Arp2/3 complex activated by WASp-WA. Tropomyosin increases the lag at the outset of polymerization, reduces the concentration of ends by 75%, and reduces the number of branches by approximately 50%. We conclude that TM bound to actin filaments inhibits their ability to act as secondary activators of nucleation by the Arp2/3 complex. This is the first example of inhibition of branching by an actin binding protein. We suggest that TM suppresses the nucleation of actin filament branches from actin filaments in the deep cortex of motile cells. Other abundant actin binding proteins may also locally regulate the branching nucleation by the Arp2/3 complex in cells.