Prime Minister Justin Trudeau says the Indian government could be behind the fatal shooting of Canadian Sikh leader Hardeep Singh Nijjar.
Mr Nijjar was shot dead outside of a Sikh temple on 18 June in British Columbia (BC).
Mr Trudeau said Canadian intelligence has identified a "credible" link between his death and the Indian state.
He raised the issue with Indian Prime Minister Narendra Modi at the recent G20 summit, he said.
"Any involvement of a foreign government in the killing of a Canadian citizen on Canadian soil is an unacceptable violation of our sovereignty," Mr Trudeau said on Monday in the House of Commons.
"It is contrary to the fundamental rules by which free, open and democratic societies conduct themselves."
India has previously denied any involvement with Mr Nijjar's murder.
Canada also expelled an Indian diplomat on Monday over the case, Canadian Foreign Minister Melanie Joly told reporters following Mr Trudeau's remarks.
The BBC has contacted the Indian embassy in Canada for comment.
Ms Joly said Canadian officials are limited in what they can say in public about the case due to the ongoing homicide investigation into Mr Nijjar's death.
Mr Nijjar was shot dead in his vehicle by two masked gunmen on a mid-June summer evening in the busy parking lot of the Guru Nanak Sikh Gurdwara in Surrey, a city about 30km (18 miles) east of Vancouver.
He was a prominent Sikh leader in the province of BC and a vocal backer of Khalistan - a separate homeland for Sikhs in the Punjab region of India. His supporters have said that he was a target of threats in the past because of his activism.
India has previously said he was a terrorist and led a militant separatist group - accusations his supporters call "unfounded".
Mr Trudeau said Canada has expressed its concerns about Mr Nijjar's death to high level security and intelligence agencies in India.
He also raised it with US President Joe Biden and UK Prime Minister Rishi Sunak.
"I continue to ask with a great deal of firmness that the government of India cooperate with Canada to shed light on this situation," he said.
Mr Trudeau's remarks come after he and Mr Modi had a tense meeting last week during the G20 summit in India.
During that meeting, Mr Modi accused Canada of not doing enough to quell "anti-India activities of extremist elements", referring to the rise of the Sikh separatist movement on Canadian soil.