The Thar Express from Jodhpur to Karachi received clearance from Pakistan for its journey on Saturday, officials said.
On Friday, Pakistan announced suspension of all train services to India, with Railway Minister Sheikh Rashid Ahmed saying in Islamabad that this would be the last Jodhpur-Karachi train.
Among the 165 passengers on board, 81 were Indians visiting relatives in Pakistan.
Eighty-four Pakistani nationals were returning to their country after their visas expired, a railway official said.
The train reached Munabao, the last station on the Indian side of the border, at 6.55 a.m. after departing from the Bhagat Ki Kothi station in Jodhpur at 1 a.m. and was waiting for clearance from Pakistan, they said.
The clearance was received at 3.05 p.m., a North Western Railway spokesperson said. The train departed soon after for the zero point on the International Border, where passengers changed trains.
The train from Pakistan reached the zero point at 10 a.m., the officials said.
The Thar Express has been running between Jodhpur and Karachi every Saturday morning since services resumed on February 18, 2006 after a 41-year suspension.
According to estimates, over four lakh passengers have taken the train in the past 13 years.
As the passengers boarded the train at Bhagat ki Kothi station, there were concerns that the journey could even end at Munabao in Barmer district, amid uncertainties over strained ties between the two countries.