"We urge you to ensure that the recently announced initiative, Start Up India, addresses the concern of Net Neutrality, with clearly defined policies and firm rules," the letter said.
Around 500 people have signed the letter including Zomato Founder Deepinder Goyal, Cleartrip Founder Hrush Bhatt, XYSEC LABS Founder Subho Halder, iSPIRT Foundation co-founder Sharad Sharma and Paytm founder Vijay Shekhar Sharma.
There are 1,467 signatories to the letter, including 681 founders of various startups.
"In the wake of the important messages made by you on helping drive forward a Startup India, and on the eve of the Republic Day celebration of our nation's commitment to democracy, we are writing to you seeking your continued support for Net Neutrality," the letter said.
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It said the open nature of the Internet allows every individual, whether online or likely to get connected to it in its current form - to be a creator and innovator by their own free will and choice.
Startups have reached out to Prime Minister close on the heels of telecom regulator Trai gearing to firm up its views on framework for differential pricing of data services.
They defined net neutrality as "Internet be maintained as an open platform, on which network providers treat all content, applications and services equally, without discrimination. This includes ensuring that network providers do not supply any competitive advantage to specific apps/services, either through pricing or quality of service."
The debate heated up after Airtel launched its free Internet platform Airtel Zero and Facebook followed suit with its Internet.Org, which was later rechristened as Free Basics.
Trai has started a public consultation on whether plans or schemes allowing differential pricing of the Internet based on its usage should be allowed or not.
This is the first consultation paper from Trai which has received maximum comments -- around 24 lakh -- till January 7.