The broadcaster said it had been pleased with Chamber's performance, despite enduring falling audience figures throughout the summer, and that he was leaving the company on good terms.
Jane Lighting, Five's chief executive, said: "Dan has done a good job at Five and has been a wonderful colleague; however, we now need a creative director with the skills and expertise to run a portfolio of channels."
Five's annual audience share has notably fallen since its high of 6.6% in 2004, and is now down 5.8% this year for the ending of the month.
Opie, who left Flextech Television in July after 13 years, worked on the creative and commercial management of various channels, including Living TV, Bravo, Trouble and Challenge. She is a former colleague of Lighting, who joined Five from Flextech where she was chief executive.
She will now be tasked with aiding Five's digital development and content strategy across its platforms, which now include its two new digital channels, Five US and Five Life, which launched two weeks ago.
Opie's hiring follows the appointment of Nick Thorogood, the former head of ITV daytime, as controller for Five's new digital channels.
Chambers is credited with bringing a stronger reality TV element to Five, but 'The Farm', 'Make Me a Supermodel' and 'Back to Reality' failed to match the success of programmes at rival channels.
He has also played a role importing a number of US dramas such as 'CSI', 'Gray's Anatomy', 'House' and 'NCIS'.
The broadcaster has experienced sliding audience figures for its crucial 8pm-11pm slot, which has so far not managed to produce an outright ratings hit.
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