Agnes began her association with the farm movement in Ontario when something she wrote for the Farmer's Sun caught the attentions of the editor, who introduced her to the United Farmer's of Ontario. She then joined the United Farm Women of Ontario but didn't limit her participation to all-female organizations, as she attended UFO meetings and helped organize farmer's clubs. Though reluctant at first, she went from only attending meetings to actually giving speeches and was elected director of the North York United Farm Women. The way she spoke her mind and feminized politics eventually brought her province-wide recognition.

In 1921, Agnes was chosen out of eleven contestants to stand as the United Farmers of Ontario candidate for the upcoming federal election. Her campaign consisted of her travelling South-East Grey by train, staying in farm houses where she could, to give speeches to voters. She stuck to her strengths. with emphasis on mistreated farmers, tariffs, education, and the flaws within Conservative government. On December 6, 1921, when she was thirty-one years old, Agnes Macphail was elected as the first female Member of Parliament with the largest majority of any Progressive Party candidate in the province.

Her first year was rough; not only did Agnes receive hostility and discrimination from her male colleagues but she was attacked by the press as well, with both male and female reporters picking at her clothes and mannerisms rather than her ideas. She became extremely discouraged and considered leaving but when her father told her that she'd proven her point and no one would blame her if she returned to teaching, Agnes vowed to go back to the House of Commons fighting.

Her political career continued and though at times Agnes felt like she would never make a difference, this was definitely not true. During the Great Depression, after seeing the destitute being mistreated by the government, Agnes helped form a new party. The Co-operative Commonwealth Federation was focused on human needs rather than making profits, embracing both workers and farmers. She went on to make strides in prison reform, better deals for workers, health services and welfare. She lost the election in 1940 but won the seat for CCF in North York in 1943 and York East in 1948. Her political career ended in 1951 but she remained an advocate for peace and equality throughout her life.