Duke is offered power armor, but he refuses the armor saying "Power armor is for pussies." This is a reference to the Halo series. It should also be noted that Halo themed music is playing in the background during the scene.

In the Duke Dome chapter before fighting the boss, Duke says "you are one ugly motherfucker." This is a reference to the movie 'Predator'.

When opening the door to Queen Bitch, Duke says "You make me wish I had three guns." This is a reference to Total Recall movie, where a women with three breasts says "you're going to wish you had three hands." Queen Bitch also has three breasts.

When in the chapter Duke's Titty City, on the desk in the Employees Only room where the security camera is, there is a never-ending spinning top. This is a reference to Inception.

In chapter Titty City when Duke interacts with a phone, he hears "Tonight... You." This is a reference to Handbanana from Aqua Teen Hunger Force.

In Duke's Titty City, when you look in the mirror in the bathroom you can see the word "MURDER" reflected off the wall (as REDRUM) in the back. This is a reference to the movie The Shining.

In Duke's Titty City there is a message "Jenny 867-5309" scribbled on the wall. It is a reference to the song "867 5309 (Jenny)" by Tommy Tutone.

In the chapter "Damn, It's late," you will have to sign a book for a kid. This book (entitled "Why I'm So Great") is the same book that Duke was being interviewed about in Duke Nukem II.

Duke was originally going to have a sidekick in the form of a gay robot. This wasn't to make fun of gay people, but to add depth to Duke's character. The idea was scrapped during development because the robot had a tragic ending, and the developers felt that Duke was more about action than tragedy.

"'Sexuality is part of the Duke personality.' The head of Gearbox explained that the original idea behind the sidekick was 'to explore how Duke would relate to a peer that might have a different sexual orientation.'"

"Ultimately the robot was in a situation where for in order for victory against the aliens to be accomplished, the robot's inner workings - a nuclear generator - had to be sacrificed."

"Duke is all about having a good time. Do you want to pay $60 to feel like crap? No!"

At one point during the game you will be accompanied by a Private Jenkins. After he runs ahead of you, he will be killed by an alien spaceship, and Duke will say, "Dammit Leroy!" This is a reference to the popular YouTube video where Leroy Jenkins gets his clan killed in World of Warcraft after charging into a dungeon screaming his name.

During the first fight with an Assault Commander boss, Duke says, "I'm going to kill you old style!" This is a reference to the Youtube video Ventrilo Harassment where a Ventrilo user is harassed by someone with a Duke Nukem soundboard. The voice actor for Duke Nukem, John St. John, has mentioned in interviews that he has seen the Ventrillo Harassment video, which probably lead to the line being in the game.

"It's awesome, especially the Ventrillo Harassment! I LOVE that fans have used my voice to create some very funny videos and soundboards."

In the level 'the Hive,' you can find the dead body of Isaac Clarke from Dead Space. Once you find him, Duke will comment, "That's one dead space marine."

In the level Blown' the Dam, there is a long Stair way with barrels are bouncing down at the player. Duke will comment, "Where's a hammer when you need one?" on the way up. At the top, you will see a Pig and Duke will say, "Huh, I was expecting a Monkey." This is an obvious reference to Donkey Kong.