Hamilton, who made his debut during the autumn internationals, has started both previous games in the 2007 tournament but has settle for a place in the 22 for the clash with the Italians.
Scotland have made two changes to their squad, with Nathan Hines, introduced as a second-half substitute in this month's victory against Wales, starting in the second-row instead of Hamilton. It will be Hines' 40th cap and he will partner Scott Murray in the second-row as Murray equals Gregor Townsend's Scotland cap record total of 82.
On the bench, Andrew Henderson replaces the injured Simon Webster.
Head coach Frank Hadden said: "Our pack stepped up to the mark against Wales and they will have to do so again as the Italians forwards are formidable.
"I couldn't really have asked for any more from Jim Hamilton in the match against the Welsh but given that Nathan (Hines) has missed out on some matches through injury I am keen to see him."
Hadden reflected that Scott Murray had marked his return to the starting line-up against Wales with a combative display. "Saturday is another landmark day for Scott and his appetite to be part of our determination to provide something special for our supporters is as strong as ever."
Some 45,200 tickets have now been sold for Saturday's match. Currently sales are averaging about 1,000 a day. Tickets costing from £10 for children and students and from £20 for adults are on sale from the Scottish Rugby Ticket Centre, Roseburn Street, Murrayfield; from the ticket hotline number 0131 346 5100; or via their website at www.scottishrugby.org. The Ticket Centre and hotline are open from 9am-5pm, Monday-Friday. Please note a booking fee applies for all credit card transactions.
Scotland team to play Italy in the RBS 6 Nations Championship at Murrayfield on Saturday February 24 (kick-off 3pm)
15 Hugo Southwell (Edinburgh Rugby)
14 Sean Lamont (Northampton Saints)
13 Marcus Di Rollo (Edinburgh Rugby)
12 Rob Dewey (Edinburgh Rugby)
11 Chris Paterson (Edinburgh Rugby) Captain
10 Phil Godman (Edinburgh Rugby)
9 Chris Cusiter (Border Reivers)*
1 Gavin Kerr (Border Reivers)
2 Dougie Hall (Edinburgh Rugby)
3 Euan Murray (Glasgow Warriors)*
4 Nathan Hines (Perpignan)
5 Scott Murray (Edinburgh Rugby)
6 Simon Taylor (Edinburgh Rugby)*
7 Kelly Brown (Border Reivers)*
8 Dave Callam (Edinburgh Rugby)*
16 Ross Ford (Border Reivers)*
17 Allan Jacobsen (Edinburgh Rugby)
18 James Hamilton (Leicester Tigers)
19 Allister Hogg (Edinburgh Rugby)*
20 Rory Lawson (Gloucester)
21 Andrew Henderson (Glasgow Warriors)
22 Nikki Walker (Ospreys)
* = Member of Scottish Institute of Sport