Major Intersections
| County | Location | Mile | Destinations | Notes |
|---|---|---|---|---|
| Sarasota | Sarasota | 0.0 | US 41 / SR 45 | Southern terminus of US 301 and SR 683 |
| 0.7 | SR 780 | |||
| North Sarasota | 4.1 | CR 610 | ||
| Manatee | Bradenton | 8.2 | SR 70 | |
| 12.0 | US 41 / SR 55 | US 301 joins US 41. SR 683 ends. | ||
| 12.6 | SR 64 | |||
| Manatee River | 13.3 | Hernando Desoto Bridge | ||
| Palmetto | 14.5 | US 41 / SR 55 / SR 43 | Diamond interchange. US 301 leaves US 41 and joins SR 43 |
|
| Ellenton | 18.0 | I-75 | Exit 224 (I-75) | |
| Parrish | 25.0 | SR 62 | ||
| 25.4 | CR 6 | |||
| Hillsborough | Sun City Center | 35.6 | SR 674 | |
| Riverview | 39.6 | CR 672 | ||
| Brandon | 49.1 | I-75 | Exit 254 (I-75) | |
| 50.6 | SR 676 | |||
| 51.1 | SR 618 | Exit 13 (FL Toll 618) | ||
| 52.5 | SR 60 | |||
| East Lake-Orient Park | 54.0 | CR 574 | Interchange with no numbers. | |
| 54.7 | SR 574 | |||
| 55.8 | I-4 / US 92 | Exit 7 (I-4) | ||
| Thonotosassa | 58.4 | CR 580 / CR 43 | SR 43 becomes Hillsborough CR 43. SR 41 begins. | |
| 60.4 | SR 582 / CR 582 | SR 582 joins US 301, then becomes CR 582 | ||
| 62.5 | CR 43 | Northern terminus of SR-CR 43 | ||
| Pasco | Zephyrhills | 73.6 | SR 56 | Future Eastern terminus |
| 74.9 | CR 535 | |||
| 75.6 | SR 39 | SR 39 becomes new hidden route. SR 41 continues | ||
| 76.8 | SR 54 | |||
| 77.7 | CR 41 | SR 41 becomes Pasco CR 41. | ||
| 78.2 | CR 54 | |||
| Clinton Heights | 83.2 | US 98 / SR 35 / SR 700 | US 98, SR 35, SR 700 join US 301. | |
| Dade City | 85.2 | SR 39 | Hidden SR 35. Former US Bus 98-301. | |
| 86.2 | SR 52 | |||
| 86.9 | SR 39 | Hidden SR 35. Former US Bus 98-301. | ||
| Trilacoochee | 92.6 | US 98 / SR 39 / SR 700 | US 98, SR 39, SR 700 leave US 301. | |
| Lacoochee | 93.3 | CR 575 / SR 575 | ||
| Withlacoochee River | 94.5 | Withlacoochee River Crossing | ||
| Hernando | Ridge Manor | 96.5 | SR 50 | |
| Sumter | Bushnell | 108 | CR 476 / CR 48 | CR 48 leaves CR 476 and joins US 301 |
| 108.5 | SR 48 | CR 48 becomes SR 48 and leaves US 301 | ||
| Sumterville | 116 | SR 471 | ||
| Wildwood | 123 | Turnpike / SR 91 | Exit 304 (Florida's Turnpike) | |
| 123.5 | SR 44 | |||
| Marion | Summerfield | 135 | Southeast 132nd Street Road | Bypass between CR 484 and US 27 / US 441. |
| Belleview | 138 | US 27 / US 441 / SR 35 / SR 500 | US 27-441-SR 500 joins US 301. SR 35 leaves US 301. |
|
| 139 | SR 25 / CR 484 | SR 25 joins US 301. Eastern terminus of CR 484. | ||
| Santos | 141 | CR 328 | Cross Florida Greenway; Santos Trailhead | |
| Ocala | 148 | SR 464 | ||
| 149 | SR 200 | Begin SR 200 multiplex | ||
| 149.5 | SR 40 | |||
| 150 | US 27 / SR 492 / SR 500 | US 27-SR 500 leaves US 301 | ||
| 151 | CR 200A | Former U.S. Route 301 Alternate | ||
| 152 | CR 25A | Former U.S. Route 441 Alternate | ||
| 154 | SR 326 | |||
| Reddick | 160 | US 441 / SR 25 | Interchange with no numbers US 441 and SR 25 leave US 301. |
|
| Citra | 165 | CR 200A | Former U.S. Route 301 Alternate | |
| Alachua | Hawthorne | 179 | SR 20 | Interchange with no numbers. |
| Orange Heights | 188 | SR 26 | ||
| Waldo | 193 | SR 24 | Interchange with no numbers. | |
| Bradford | Starke | 205 | SR 100 | |
| 205.5 | SR 230 | |||
| 206 | SR 16 | |||
| Duval | Maxville | 224 | CR 228 | County extension of SR 228 |
| Baldwin | 230 | I-10 / SR 8 | Exit 343 (I-10) | |
| US 90 west / SR 10 | South end of US 90 overlap | |||
| US 90 east / SR 10 | North end of US 90 overlap | |||
| Nassau | Callahan | 252 | US 1 / US 23 / SR 15 / SR A1A | End SR 200 multiplex Begin US 1-23 multiplex |
| Hilliard | 263 | CR 108 | ||
| Boulogne | 270 | CR 121 | Not part of SR 121 | |
| St. Mary's River | 270.5 | St. Mary's River Crossing. Florida-Georgia State Line | ||
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