Route 301 or Highway 301 could refer to:

Canada

• Manitoba Provincial Road 301
• Newfoundland and Labrador Route 301
• Nova Scotia Route 301
• Prince Edward Island Route 301
• Quebec Route 301
• Saskatchewan Highway 301

China

• China National Highway 301

Costa Rica

• National Route 301

Japan

• Japan National Route 301

Korea, South

• Sangju–Yeongcheon Expressway

Philippines

• N301 highway (Philippines)

United States

• U.S. Route 301 (former)
• U.S. Route 301
• Arkansas Highway 301
• Georgia State Route 301
• Indiana State Road 301
• Iowa Highway 301 (former)
• Kentucky Route 301
• Louisiana Highway 301
• Maryland Route 301
• Minnesota State Highway 301
• Mississippi Highway 301
• New York:
  • New York State Route 301
  • County Route 301 (Albany County, New York)
  • County Route 301 (Erie County, New York)
  • County Route 301 (Westchester County, New York)
• North Carolina Highway 301 (former)
• Ohio State Route 301
• Pennsylvania Route 301 (former)
• Tennessee State Route 301
• Texas:
  • Texas State Highway 301 (former)
  • Texas State Highway Loop 301
  • Farm to Market Road 301
• Utah State Route 301
• Virginia State Route 301

Other areas:

• Puerto Rico Highway 301
• U.S. Virgin Islands Highway 301

Film

• Highway 301 (film), a 1950 film noir by Andrew L. Stone