Malaysian Prime Minister Najib Razak and his Singapore Counterpart Lee Hsien Loong witnessed the signing of the 350-km project agreement here.
Apart from reducing travel time between the countries to just 90 minutes, the project will enhance business ties and improve people-to-people relations.
The high-speed rail line, first mooted in 2013, is billed as a "game changer" for both countries.
Earlier the two leaders met one-on-one followed by a delegation level meeting where both sides reviewed progress of existing bilateral cooperation and building on the momentum of discussion held and initiatives agreed during the 2015 Leaders Retreat.
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Customs, Immigration and Quarantine (CIQ) facilities will also be co-located at Singapore, Bandar Malaysia and Iskandar Puteri. This means that international passengers will only need to undergo both Singapore and Malaysia CIQ clearance at the point of departure.
It will have eight stations between Singapore and Malaysia with the two terminal stations at Jurong East in Singapore and Bandar Malaysia in Kuala Lumpur.
In 2015, Singapore was Malaysias second largest trading partner globally.
Last year, total trade between the two countries was recorded at USD 59.5 billion (RM190.6 billion).