After the opening round loss to Doncaster, Ayr are boosted by the return of Frazier Climo to the No.10 slot while Glasgow pros Ed Kalman and Pat MacArthur start with Hamish Mitchell on the bench.
Ayr also list Glasgow Hawks' back row man Greg Francis among the replacements.
Rotherham include former Glasgow pros Gregor Hayter and Ben Prescott.
Ayr skipper Damien Kelly said: "The crowd are always fantastic at Millbrae and we know we can't let them down and since we have been termed a big game team in the past, we should perform since club games don't come much bigger than this one."
Tickets, available on the gate, are priced at £10 for adults and £5 for OAPs. Under 18s are free
15 Grant Anderson
14 A Wilson
13 Ross Curle
12 Mark Stewart
11 Steven Manning
10 Frazier Climo
9 Jamie Hunter
1 Gordon reid
2 Pat MacArthur
3 Ed Kalman
4 Scott Sutherland
5 Damien Kelly CAPTAIN
6 Andy Dunlop
7 Paul Burke
8 Glen Tippett
Replacements
16 Hamish Mitchell FR
17 Stewart Fenwick FR
18 Gordon Sykes FR
19 Scott Nimmo
20 Greg Francis
21 AJ MacFarlane
22 Dougie Steele