As heir to the throne, Guillaume had taken a much keener interest in Luxembourg than his father. He had Berg Castle rebuilt and made it his residence. On 17 November 1905, Prince Guillaume succeeded his father as Grand Duke of Luxembourg. In his accessional speech, he confirmed an intention to remain above and outside of partisan politics.
With no male heir to succeed him, Grand Duke Guillaume promulgated a new family statute permitting females to succeed to the throne. The statute was approved by the Chamber of Deputies on 16 April 1907 and, accordingly, Princess Marie-Adelaide became heir to the throne.
Grand Duke Guillaume was plagued by ill health for much of his reign. In 1908 he appointed his wife, Grand Duchess Marie-Anne, as his Lieutenant-Representative and, later that same year, she took the constitutional oath as Regent of the Grand Duchy.
Grand Duke Guillaume died on 25 February 1912 at Berg Castle. Grand Duchess Marie-Anne survived him and died on 31 July 1942 in New York, whilst in exile from the German invasion. In 1947, her body was returned to Luxembourg and laid to rest alongside her husband in the Cathedral of Our Lady.
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