The actress has revealed that she will not be returning to the role of Peggy Carter, reports Time Out.
Speculation as to who will play the female lead - suspected to be Sharon Carter - was rife but has fallen silent in recent months.
Jessica Brown Findlay (Downton Abbey), Emilia Clarke (Game of Thrones), Teresa Palmer (I am Number Four), Imogen Poots (Fright Night) and Alison Brie (The Five-Year Engagement) were rumoured to be in the running for the part.
Chris Evans will reprise his starring role in the sequel, which will be set in the present day.
Joining him will be newcomers Anthony Mackie as the Falcon and Frank Grillo as Crossbones, with Samuel L Jackson, Scarlett Johansson and Cobie Smulders reprising their roles from previous Marvel Studios films.
The Anthony and Joe Russo-directed Captain America: The Winter Soldier will be released on April 4, 2014.