The Brazilian actor will reprise his role as Xerxes in the Warner Bros film, which takes place at the same time as Zack Snyder's 2008 movie.
"I had to shave, I had to work out again, I had to go back to Xerxes," he told ComingSoon.net.
"It's the same character. There's little new things - a little before, a little after - but most of the movie, it's during... kind of the same time where the first movie takes place.
"There's a lot going on. It's hard, the green screen, it's a different way of working. It's really challenging to work that way and the character wears a lot and Xerxes is a giant. He's almost not human, which is a poetic licence.
"It comes from a graphic novel so it's not based in real history, so that character is very, very tall and his voice is like thunder and that's the description of this character. So there's a couple of special FX and tricks that have to be done to accomplish that figure so I do scenes by myself. It's interesting, it's a lot of work."
Noam Murro is directing the film, with Kurt Johnstad joining Snyder to work on the script.
Sullivan Stapleton, Eva Green, Lena Headey, Hans Matheson, Yigal Naor, Callan Mulvey, Jack O'Connell and Andrew Tiernan will also star.
Santoro will next be seen in The Last Stand alongside Arnold Schwarzenegger.
300: Rise of an Empire is yet to have a release date set.