The member for Euroa, who's expecting her second child, will step away at the November state election after eight years.
"With a little one at home and another on the way, it is time for me to seek a job that offers greater flexibility," she said in a statement.
"Serving my community as the first member for Euroa has been the honour of a lifetime.
"The greatest pleasure of this job has been the opportunity it has afforded me to meet everyday people doing extraordinary things."
Victorian Nationals leader Peter Walsh said Ms Ryan, 36, had been a "driving force for positive change for regional Victorians".
"Steph leaves this role, and her roles in the shadow cabinet, with a proud record of championing a better future for country people where it's needed most," he said.
In 2014, Ms Ryan became the first woman to hold a leadership position in the Victorian National Party and won her seat with almost 65 per cent of the vote.
She is married to former Labor City of Yarra Councillor Simon Huggins, with whom she has a daughter.
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