On 1 September 1939 the regiment was mobilized as part of the Canadian Active Service Force as Canada prepared for participation in the Second World War. When war was declared on September 10, the RCR had already been allocated to the 1st Canadian Infantry Brigade, a formation made up entirely of Ontario units. Moving to the United Kingdom in December 1939 as a component of the 1st Canadian Infantry Division, the RCR saw hard training for almost four years.

On 10 July 1943, the RCR landed at Pachino in the opening waves of the Allied invasion of Sicily – the last member of the regiment to participate in these original waves – George F. Burrows of Chatham Ontario – died on April 11, 2012, in Windsor, Ontario at the age of 88. The regiment and its sister units in the 1st Brigade, The Hastings and Prince Edward Regiment and the 48th Highlanders of Canada fought in several battles as the division advanced north and then east towards Messina. After the 38-day campaign on the island was completed, the regiment was involved in another amphibious landing (codenamed Operation Baytown) at Reggio di Calabria, as part of Allied invasion of the Italian mainland in September.

The RCR fought in several battles of the Italian campaign, including key engagements in the Moro River valley near Ortona in December 1943. During 1944, the regiment took part the Battle of Monte Cassino in attacks on German defensive lines called the Hitler Line and later the Gothic Line.

The regiment was transferred to northwest Europe in February 1945 during Operation Goldflake and took part in the liberation the Dutch city of Apeldoorn. The regiment received 28 battle honours for its participation in the Second World War. The regiment returned home to Canada in 1945.