MILAN, May 27 (Reuters) - Francesco Gaetano Caltagirone, the second largest shareholder in Assicurazioni Generali, has quit its board, the Italian insurer said on Friday, opening a new chapter in a long-standing shareholder row.

Caltagirone holds a 9.95% stake in Generali, having beefed up his holding in recent years as tensions mounted between the construction and newspaper magnate on the one hand, and the insurer's board and its single biggest investor Mediobanca on the other.

Shares in Generali fell more than 2% after the news, underperforming a flat Italian blue-chip index on investor concerns that Caltagirone might reduce his stake.

However, a source close to the matter told Reuters the holding remained strategic and Caltagirone had no plans to sell it.

Caltagirone earlier this year challenged a decision by Generali's board to secure a third term for CEO Philippe Donnet but his bid to appoint his own CEO candidate was defeated in a shareholder vote last month.

Caltagirone did secure three board seats for his list of nominees at the general meeting on April 29 and had taken up one of these.

Generali said it would call a board meeting in the coming days to take a decision on Caltagirone's replacement, adding the reasons behind his decision were not known.

A person with knowledge of the matter said Caltagirone had informed Generali of his decision in a letter on Friday.

Caltagirone stepped down from the previous Generali board in January before proposing a rival to Donnet for the top job.

(Reporting by Gianluca Semeraro and Stefano Bernabei; Writing by Valentina Za, editing by Giulia Segreti and Keith Weir)