His most noted role was as B-52 pilot Major T. J. "King" Kong in Dr. Strangelove, which ended with Pickens riding an H-bomb down to global destruction, while waving his hat. He was chosen because he naturally fit the role of an absurdly patriotic and gung-ho cowboy-type officer, and in fact was not even told that the movie was a comedy: he was instructed to play the role straight, and was only given script portions for the scenes he was in, rather than a script for the whole film. He is best known for both the scene riding the bomb to destruction, and his over-the-top speech early in the film, after his character learns of the mission to bomb strategic targets within Russia:
"Well, boys, I reckon this is it — nuclear combat toe to toe with the Rooskies. Now look, boys, I ain't much of a hand at makin' speeches, but I got a pretty fair idea that something doggone important is goin' on back there. And I got a fair idea the kinda personal emotions that some of you fellas may be thinkin.' Heck, I reckon you wouldn't even be human bein's if you didn't have some pretty strong personal feelin's about nuclear combat. I want you to remember one thing, the folks back home is a-countin' on you and by golly, we ain't about to let 'em down. I tell you something else, if this thing turns out to be half as important as I figure it just might be, I'd say that you're all in line for some important promotions and personal citations when this thing's over with. That goes for ever' last one of you regardless of your race, color or your creed. Now let's get this thing on the hump — we got some flyin' to do."
Pickens credited the film as turning point in his career, saying "After 'Dr. Strangelove,' the roles, the dressing rooms, and the checks all started gettin' bigger." He also claimed that before 'Dr. Strangelove' he was "HEY YOU" on sets, and after it, he was called "Mr. Pickens." He seemed amazed at such a change occurring after one movie.

Pickens appeared in dozens of films, including, Old Oklahoma Plains (1952), Down Laredo Way (1953), One-Eyed Jacks (1961) with Marlon Brando, Major Dundee (1965) with Charlton Heston, the remake of Stagecoach (1966; Pickens played the driver, portrayed in the 1939 film by Andy Devine), The Cowboys (1972) with John Wayne, Ginger in the Morning (1974) with Fred Ward, Blazing Saddles, Poor Pretty Eddy (1975), Rancho Deluxe (1975), The Getaway (1972) with Steve McQueen, Tom Horn (1980) also with Steve McQueen, and Pat Garrett & Billy the Kid (1973) in a small but memorable and moving role. He also had a small role in Steven Spielberg's 1941 (1979) in scenes with Toshiro Mifune and Christopher Lee. In 1978, Pickens lent his voice to theme park Silver Dollar City as a character named Rube Dugan for a ride called "Rube Dugan's Diving Bell". The Diving Bell was a simulation ride that took passengers on a journey to the bottom of Lake Silver and back. The ride was in operation from 1978 to 1984.

Pickens was offered the part of Dick Hallorann in Stanley Kubrick's 1980 adaptation of Stephen King's The Shining. He refused, saying that filming with Kubrick on Dr. Strangelove was too strenuous. He later relented, saying that he would appear in the film as long as Kubrick was contractually required to shoot Pickens' scenes in fewer than 100 takes a shot. However, the role eventually went to Scatman Crothers.

He also appeared many times on television, both in guest shots, and in regular roles in The Legend of Custer, Bonanza, Hee Haw, B.J. and the Bear, and Filthy Rich (1982). He played the owner of station WJM, Wild Jack Monroe, on the Mary Tyler Moore Show.