Nope.avi is a video on youtube made in Source Filmmaker wherein a BLU Engineer runs up to the camera from a tunnel in the background. His helmet rises up off of his head once he reaches the foreground. He then says "Nope" before his head rises up back into his helmet causing his neck to stretch before he moves on.
Over the time since its creation, it has achieved a kind of memetic status and is frequently posted instead of straight-up saying "no" in response to an objectionable statement. It has also been repeatedly parodied in various photoshops.