The vote was 52-48. It marked the first time in U.S. history the Senate confirmed an openly transgender federal official.
Senate Majority Leader Chuck Schumer, D-N.Y., called the confirmation vote another significant "milestone" after Transportation Secretary Pete Buttigieg became the first openly gay Cabinet member to be confirmed by the Senate in February.
"The Biden administration has brought many historic firsts into its ranks," Schumer said Wednesday prior to the vote. "... The confirmation of Rachel Levine represents another important milestone for the American LGBTQ community. She will be the first openly transgender official ever confirmed by the United States Senate. The arc of history is long, but it keeps bending in the direction of justice."
Levine will serve under newly confirmed Health and Human Services Secretary Xavier Becerra.
In addition to handling medical, public health and social services programs, the HHS department is responsible for sheltering the influx of unaccompanied migrant children crossing into the United States once they are released from border patrol custody.
During her confirmation hearing, some Republicans raised concerns about Levine's handling of the coronavirus response in Pennsylvania, including nursing home deaths, and cast doubt on whether she deserves a promotion.
Sen. Richard Burr, R-N.C., told Levine: "Along with the testing challenges from last spring, your state failed to adequately protect nursing home residents from the virus, and is making unacceptable mistakes in the vaccine distribution process. Pennsylvania ranks as one of the most dangerous states for long-term care residents battling COVID-19."
Sen. Rand Paul, R-Ky., pressed Levine on where she stands on "genital mutilation" of minors who may be "confused" about gender identity and want to take hormones or have surgery to alter their body.
Levine evaded a direct answer and responded: "Transgender medicine is a very complex and nuanced field with robust research and standards of care that have been developed."
Democrats panned Paul for his line of questioning.
Meanwhile, Schumer praised Levine's qualifications for the job and said she's stayed "laser-focused" on responding to the coronavirus pandemic in Pennslyvania "despite several attacks on her gender identity over the past year."
Schumer said he hoped Levine's prominent position in the Biden administration will further break down stereotypes.
"As transgender Americans suffer higher rates of abuse, homelessness and depression than almost every other group, it's important to have national figures like Dr. Levine, who by virtue of being in the public spotlight, will help break down barriers of ignorance and fear," Schumer said.
A Harvard-educated pediatrician and former Pennsylvania physician general, Levine was appointed to be Pennsylvania Health Secretary by Democratic Gov. Tom Wolf in 2017, making her one of the few transgender people serving in elected or appointed positions nationwide. She is a professor in pediatrics at the Penn State College of Medicine.
In 2020, Levine became president of the Association of State and Territorial Health Officials (ASTHO).
She won past confirmation by the Republican-majority Pennsylvania Senate and was the public face of the state's response to the coronavirus pandemic.
The Associated Press contributed to this report.