Prague and Taipei signed a partnership agreement on Monday in an apparent snub to Beijing.
Prague had dropped Beijing as a sister city just three months ago.
The move is expected to revive an ongoing dispute between the Czech and Chinese capitals that has soured Czech-Chinese relations despite a campaign by Czech president Milos Zeman for closer ties.
Prague mayor Zdenek Hrib, from the anti-establishment Pirate Party, cancelled a twinning agreement with Beijing in October in protest at Chinese insistence on a one-China policy.
Hrib hailed the new twinning with Taipei as “most beneficial” for both parties on Monday, citing “shared democratic values, respect for fundamental human rights and cultural freedoms.”
Taiwan has been ruled separately from China since the end of a civil war in 1949, but under its “One-China” policy, Beijing considers it a part of its territory, with reunification by force an option.
“Prague has its own choice to become a sister city with cities of the world and I think Beijing should also let Prague the right to choose,” Taipei Mayor Ko Wen-je told AFP after Monday’s ceremony, speaking via an interpreter.
Hrib condemned China as an “unreliable partner” in an interview run by German newspaper Welt am Sonntag on Sunday.
He added China was “full of resentment” and was trying to influence Czech public opinion, and that he could not sign an agreement that forced Prague to “speak out against the independence of Tibet and Taiwan.”
Monday’s signature comes only days after Taiwan emphatically re-elected incumbent President Tsai Ing-wen, a result widely seen as a rebuff to China.
Hrib also accused the Czech government of “neglecting” ideals of the peaceful 1989 Velvet Revolution that ended four decades of communist rule in former Czechoslovakia, as it bows to China on many fronts.
President Zeman is well known for his pro-Chinese stance, while the financial PPF Group of the wealthiest Czech, Petr Kellner, is a major player on China’s loan market.