Finding a simple fabfile with a working example of SSH keyfile usage isn't easy for some reason. I wrote a blog post about it (with a matching gist).

Basically, the usage goes something like this:

from fabric.api import *

env.hosts = ['']
env.user = 'user'
env.key_filename = '/path/to/keyfile.pem'

def local_uname():
    local('uname -a')

def remote_uname():
    run('uname -a')

The important part is setting the env.key_filename environment variable, so that the Paramiko configuration can look for it when connecting.