"Time to draw a line in the sand, Cleartrip is pulling out of Internet.org and standing up for #NetNeutrality," Cleartrip said on microblogging site Twitter.
Details of the decision to withdraw from Internet.org, which was launched in February this year in India, could not come through as an e-mail query went unanswered.
Earlier in the day, Facebook chief Mark Zuckerberg rejected criticism that the internet.org programme, which has RCom as a partner in India, was against the concept of Net neutrality.
"For people who are not on the Internet though, having some connectivity and some ability to share is always much better than having no ability to connect and share at all. That's why programmes like internet.org are important and can co-exist with Net neutrality regulations," he said.
internet.org is a Facebook-led initiative which states that it aims to bring 5 billion people online in partnership with tech giants like Samsung and Qualcomm.
Facebook's partnership with Reliance Communications to provide free Internet access to 33 websites as part of its internet.org initiative has raised eyebrows, with free Internet activists saying it violates the idea of Net neutrality.
The debate in India has been triggered by mobile operator Airtel introducing an open marketing platform, Airtel Zero, and TRAI's consultation paper on whether telecom firms can be allowed to charge different rates for Internet data like e-mail, browsing and use of apps like Whatsapp, Viber and Sky.
Net neutrality implies equal treatment for all Internet traffic and no priority given to an entity or company based on payment to service providers like telecom companies, which is seen as discriminatory.
Yesterday, Flipkart founder Sachin Bansal said: "...after looking in deeper, we realised that Net neutrality can get compromised in future, which we are not supportive of at all."