It was the England all-rounder's first appearance for his county since being ruled out with a side strain at the beginning of May, but he was out first ball to Tim Bresnan as the hosts lost by four runs.
Yorkshire made just 135 for eight.
England Test skipper Michael Vaughan also endured a miserable night when he was dismissed by international colleague Sajid Mahmood for nought to reduce Yorkshire to eight for two at one stage.
Lancashire's response saw Stuart Law fall for one at the hands of Bresnan to herald the arrival at the crease of Flintoff.
England bowler Matthew Hoggard then removed Mal Loye for 17 as Lancashire struggled to 20 for three.
The hosts entered the final over needing 12 and six off the last ball but could only manage one as Yorkshire claimed a four-run victory.