Earlier this month Italy said it would delay the loan to Israel of "The Annunciation of San Martino alla Scala" with the culture ministry in Rome saying the timing was "not appropriate".
Italy's decision coincided with regional tensions that soared in anticipation of possible US military action in Syria, now prevented following Saturday's US-Russian deal on Syria's chemical arsenal.
Then earlier this week the museum announced that the Botticelli - the first to go on display in Israel - would be arriving on schedule.
A spokesman for Israeli Culture Minister Limor Livnat said she did all she could to convince her Italian counterpart that "there was no danger in bringing the artwork, and the Israel Museum had all the necessary conditions to safeguard it."
According to a museum spokeswoman, the near-delay was not due to regional tensions but rather a matter of conservation.
Painted in 1481, the work was intended for the hospital of San Martino alla Scala in Florence.
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After suffering heavy damage, it was transferred to a Florentine workshop in 1920 for restoration.
Speaking at Tuesday's opening, Israel Museum director James Snyder said the fresco had a deep connection to the Holy Land since the annunciation story took place in Nazareth, and "the landscape in the fresco is the landscape of this ancient land.