In a surprising move, Greg Burke wrote on Twitter that it was the right time for the Pope to put together a new team.
His deputy, Paloma Garcia Ovejero, has also stepped down.
The sudden departures come amid some structural changes to the Vatican's media operations.
Pope Francis has moved quickly to appoint Alessandro Gisotti, a current member of the Vatican's media office, as an interim replacement.
Making the announcement, Greg Burke said: "I joined the Vatican in 2012. The experience has been fascinating, to say the least. Thank you, Pope Francis.
"Paloma and I have resigned, effective Jan. 1. At this time of transition in Vatican communications, we think it's best the Holy Father is completely free to assemble a new team.
"New Year, New Adventures."
Burke previously worked as a correspondent for Fox News.
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