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 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, Pavan Kumar Rai, on Monday over the case, 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.
Investigators have previously categorised the 45-year-old's death as a "targeted incident".
Mr Nijjar was shot dead in his vehicle by two masked gunmen on a mid-June summer evening in the busy car park 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 western-most province of British Columbia and publicly campaigned for Khalistan - an independent Sikh homeland 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 described him as a terrorist who 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 co-operate with Canada to shed light on this situation," he said.
Mr Trudeau said that Mr Nijjar's shooting has angered Canadians, leaving some fearful for their safety.
Some Sikh groups in Canada, including the World Sikh Organisation, welcomed the prime minister's statement, saying Mr Trudeau confirmed what was already widely believed in the community.
There are an estimated 1.4 to 1.8 million Canadians of Indian origin. The country has the largest population of Sikhs outside the state of Punjab in India.
Mr Trudeau's remarks come after his tense meeting with Mr Modi last week during the G20 summit in India.
During that meeting, according to a statement from the Indian government, Mr Modi accused Canada of not doing enough to quell "anti-India activities of extremist elements", referring to the Sikh separatist movement in the country.
Canada also recently suspended negotiations for a free trade agreement with India. It gave few details on why, but India cited "certain political developments".
Mr Nijjar is the third prominent Sikh figure to have died unexpectedly in recent months.
In the UK, Avtar Singh Khanda, who was said to be the head of the Khalistan Liberation Force, died in Birmingham in June under what has been described as "mysterious circumstances".
Paramjit Singh Panjwar, who was designated a terrorist by India, was shot dead in May in Lahore, the capital of Pakistan's Punjab province. (BBC)