The European Union's Court of Justice upheld a lower court's decision, dismissing the company's appeal against the 2.4 billion euro ($A4 billion) penalty from the European Commission, the 27-nation bloc's top antitrust enforcer.
"By today's judgment, the Court of Justice dismisses the appeal and thus upholds the judgment of the General Court," the court said in a statement on Tuesday.
The commission's original decision in 2017 accused the Silicon Valley giant of unfairly directing visitors to its own Google Shopping service to the detriment of competitors.
It was one of three multibillion-euro fines that the commission imposed on Google in the previous decade as Brussels started ramping up its crackdown on the tech industry.
Google made changes to comply with the commission's decision requiring it to treat competitors equally.
The company started holding auctions for shopping search listings that it would bid for alongside other comparison shopping services.
At the same time, the company appealed the decision to the courts.
But the EU General Court, the tribunal's lower section, rejected its challenge in 2021 and the Court of Justice's adviser later recommended rejecting the appeal.
Google is also appealing the other two EU antitrust penalties involving its Android mobile operating system and AdSense advertising platform.
The company was dealt a setback in the Android case when the EU General Court upheld the commission's 4.125 billion euro fine in a 2022 decision.
Its initial appeal against a 1.49 billion euro fine in the AdSense case has yet to be decided.
Those three cases foreshadowed expanded efforts by regulators worldwide to crack down on the tech industry.
The EU has since opened more investigations into tech companies and drafted new laws to clean up social media platforms and regulate artificial intelligence.
Google is facing particular pressure over its digital advertising business. In a federal antitrust trial set to begin on Monday, the US Department of Justice alleges the company holds a monopoly in the "ad tech" industry.
British competition regulators accused Google last week of abusing its dominance in "ad tech" while the EU is carrying out its own investigation.
