Napthine was elected to the Victorian Legislative Assembly in 1988 as member for the country seat of Portland. He was re-elected in 1992, 1996 and 1999. Napthine was appointed Parliamentary Secretary to the Minister for Health in 1992 and then promoted to Minister for Youth and Community Services in 1996 in Jeff Kennett's Liberal government, a position he held from 1996 to 1999. Following the defeat of the Liberal Party at the 1999 election, Kennett resigned as Liberal leader and Napthine was elected to take his place. Napthine had briefly served as Deputy Liberal Leader, having been elected to the position just shortly before Kennett stood down as Liberal Leader. During his term as Opposition Leader the Liberal–National coalition split, fracturing the opposition parties. Under Napthine's leadership, the Liberal Party did poorly in the polls and made no significant electoral inroads on the ruling Labor government. Shortly before the 2002 state election, Shadow Health Minister Robert Doyle challenged Napthine for the leadership of the Liberal Party. Doyle won the leadership by a narrow margin. At this election, Napthine ran in South-West Coast, essentially a reconfigured version of Portland. Although it was ancestrally Liberal territory, Napthine only managed to hold onto the seat with a significantly reduced margin which fell from 4.9 to only 0.7 points. He was re-elected in November 2006 with an increased margin. After the Liberals won the 2010 Victorian election, Napthine was made a member of the Baillieu cabinet. He served as the Minister for Ports, Racing, Regional Cities and Major Projects.
Premier of Victoria
On 6 March 2013, Baillieu resigned as state leader of the Liberal Party and hence as Premier of Victoria. At an emergency meeting of the Liberal partyroom, Napthine—who had turned 61 that day—was elected his successor. After the resignation of Geoff Shaw from the parliamentary Liberal Party earlier in the day, Napthine began leadership of a minority government, holding 43 seats to Labor's 43 seats, with Shaw sitting as an independent politician on the crossbench. On 28 November 2013Australian media reported that Napthine had 'secretly assisted' Shaw to draft a Private Member's Bill to change state abortion legislation which Napthine voted against in 2008. Napthine refuted the allegations and stated his Government had no plans to change abortion laws, nor would he support any such change. In June 2014 Napthine stated:
"...that while I am Premier of this state, I will not allow Mr Shaw to introduce any legislation seeking to change the abortion laws in Victoria."
During September 2014 Napthine and Daniel Andrews told the Australian Christian Lobby that they would allow a conscience vote should a private members bill to repeal section eight of the Victorian abortion laws be introduced. Section eight if removed would mean doctors are not required by law to refer a woman who wants an abortion to an abortion doctor or perform the abortion even if they have a conscientious objection. In May 2014 a parliamentary privileges committee found that Shaw was not diligent in the use of his parliamentary car and fuel card. On 3 June 2014 Shaw told radio station 774 ABC Melbourne that he would support a no confidence motion in the Napthine government. On 11 June 2014, following a Labor move to expel Shaw from Parliament that was defeated on party lines with the support of the speaker, Napthine's government successfully moved a motion that resulted in the Members of the Legislative Assembly suspending Shaw for a period of eleven days, fined and ordered to apologise to the Parliament upon his return. Shaw's suspension followed a period of political instability under Napthine's leadership.
In opposition
On 29 November 2014, after the Victorian state election, Napthine conceded defeat to Labor led by Daniel Andrews. Napthine stated that he intended to serve a full parliamentary term despite losing the Premiership, however on 31 August 2015 he announced his resignation from the Victorian Parliament, which triggered a by-election in his electorate of South-West Coast.