At least 400 Nepalese protesters blocked major trading checkpoints between Nepal and India on Friday.
Tensions have risen in the country over a new constitution, leading to often violent protests.
The protesters have cut off more than 1,000 oil tankers and trucks with vital supplies for the landlocked Himalayan country.
The new constitution, which Nepal's parliament adopted on Sunday, mandates the division of the country into seven federal provinces.
Protests are being held mainly in the Madhesi and Tharu regions and more than 40 people have died in clashes with police.
The protesters, who mostly belong to ethnic minorities, say the new internal borders leave them underrepresented in the national parliament.
Shiva Patel, general secretary of the regional Sadbhawana political party, told AFP "We blocked the crossing overnight and we will not budge until the government listens to us and makes changes to these new borders in the constitution.”
One hundred and thirty five kilometers south of Nepal’s capital Kathmandu in Birgunj, a town on the border with India which is a key center for oil and food imports into the country, checkpoints have been blocked by protesters.
About 1,500 oil tankers from India used to enter southern Nepal every day as well as trucks carrying sugar, salt, food and cooking gas cylinders.
"Normally hundreds of trucks would pass through this checkpoint overnight but not even one turned up last night," Patel told AFP.
"The blockade is our last resort to make the government understand our demands," he added.
India has accused the Nepalese government of being responsible because of Nepal allows the trade trucks to travel unescorted.
"Our freight forwarders and transporters... [have] voiced complaints about the difficulties they are facing in movement within Nepal and their security fears, due to the prevailing unrest," said New Delhi in a statement released Friday.
"Nepalese leadership needs to address the causes underlying the present state of confrontation credibly and effectively. Issues of differences should be resolved."
Shishir Dhungana, the director-general of customs in Nepal, said that toughened Indian security measures are also slowing down trade.
"Things have slowed down because Indian customs and security officials are carrying out more checks than usual," said.
Nepal's Prime Minister Sushil Koirala cancelled his scheduled the US on Wednesday.
Maoist former Prime Minister Baburam Bhattarai of the country's said that he was supporting the protesters and criticised the government for not taking their concerns into consideration when they were drafting the constitution.
"I urge the government to create an environment so meaningful talks can take place," Bhattarai said in a statement released late Thursday.
The drafting of Nepal’s new constitution started in 2008 after Maoist rebel groups laid down arms in 2006.
In 2008 Maoists won parliamentary elections in Nepal and ended the country's monarchy, but power-sharing squabbles between parties stymied progress on the draft.