The Saudi Arabian foreign ministry has recalled its ambassador from Canada and given the Canadian ambassador 24 hours to leave the country.
The move comes after Canada said it was "gravely concerned" about the arrest of several human rights activists.
The dispute appears centered around tweets by Canadian diplomats calling on the kingdom to "immediately release" women's rights activists recently detained by the kingdom.
It said it will not accept any form of interference in its internal affairs and considers the Canadian position "an attack" requiring a firm stance to deter "attempts to undermine the sovereignty" of Saudi Arabia.
Those rounded up in recent days include Samar Badawi, who is known for having challenged the kingdom's male guardianship rules and is the sister of one of Saudi Arabia's most prominent detainees, liberal blogger Raif Badawi.
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Riyadh said Canada's statement about "civil society activists" was negative and unfair to the truth.
The Saudi foreign ministry slammed the Canadian statement, signalling its growing irritation over Western criticism of the kingdom's poor human rights record.
"We urge the Saudi authorities to immediately release them and all other peaceful human rights activists", Global Affairs Canada said on its Twitter feed. Bilateral trade between the two nations reached $3 billion in 2016, with tanks and fighting vehicles among the top Canadian exports to the kingdom, according to government statistics.
Badawi's wife Ensaf Haidar has been granted asylum by Canada, where she is raising their three children now aged 14, 13 and 10 as a single mother.
The Trudeau government has taken a lot of criticism for issuing export permits for a deal to sell billions of dollars of weaponized armoured vehicles to Saudi Arabia, a country that ranks among the "worst of the worst" on human rights by US watchdog Freedom House. "The persons referred to were lawfully detained by the public prosecution for committing crimes punishable by applicable law, which also guaranteed the detainees' rights and provided them with due process during the investigation and trial".
In April, Canadian Prime Minister Justin Trudeau expressed his "serious concern" over the continued jailing of Badawi to Saudi King Salman.