Nakajima's 1:17.494 lap was nearly four tenths of a second quicker than the previous best, set by Brawn GP's Jenson Button on Tuesday.
The Williams FW31's strong pace added more intrigue ahead of the start of the season in Melbourne in less than two weeks' time.
Nakajima finished ahead of McLaren's Heikki Kovalainen, who set the British squad's best time of the week after 84 laps. The Finn's day ended a few minutes early after his car stopped on track.
With in-season testing banned this year, Jerez's test was the final one for several months.
Pos Driver Team Time Laps
1. Nakajima Williams-Toyota (B) 1:17.494 102
2. Kovalainen McLaren-Mercedes (B) 1:17.946 84