Samantha Cohen, 50, will leave her role after the birth of their child in spring.
Confirmation that she is leaving – revealed by The Times – comes just weeks after another aide suddenly resigned amid rumours that the Duchess is difficult to work for.
Ms Cohen – nicknamed 'Samantha the Panther' for her feisty attitude – used to be one of The Queen's most trusted aides before moving to work for Meghan and Harry.
She has served as a communications secretary, and has spent 17 years working for the royal family.
One of her key roles was said to be training Meghan in royal etiquette.
The Australian, who is a mother-of-three, was filling a role left vacant by Edward Lane Fox, and Harry and Meghan reportedly wanted her to stay on in the role.
A source close to Meghan said: "Sam will be a huge loss.
"Going forward, Meghan might need someone cut from a slightly different cloth to traditional courtiers, who is not a career civil servant or royal insider."
Another high profile aide for Meghan, Melissa Toubati, 39, sensationally resigned just six months after the royal wedding.
The French assistant who played a "pivotal role in the success of the Royal Wedding" ditched her appointment after "putting up with a lot".
A source told the Mirror: “Her job was highly pressurised and in the end it became too much.
"She put up with quite a lot. Meghan put a lot of demands on her and it ended up with her in tears.
“She is hugely talented and played a pivotal role in the success of the royal wedding. She’ll be missed by everyone in the household.
"Melissa is a total professional and fantastic at her job, but things came to a head and it was easier for them both to go their separate ways.”
The PA had previously worked "happily" for Robbie Williams.
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