Cowboys defensive end DeMarcus Lawrence had his appeal denied and has been suspended for the first four games of the season for violating the NFL's substance abuse policy.
Lawrence, who led the team with eight sacks last season, is being disciplined for using amphetamines, sources told ESPN in April.
This is the second consecutive year the Cowboys will start the season without at least two key defensive players. Second-year defensive end Randy Gregory will miss the first four games of the season for violating the league's substance abuse policy, and as also reported Thursday, middle linebacker Rolando McClain was suspended the first 10 games for violating the substance abuse policy.
McClain (substance abuse) and defensive end Greg Hardy (personal conduct) were both suspended for the first four games of the 2015 season.
Lawrence will be eligible to return to the roster on Oct. 3, and is eligible to participate in all preseason practices and games.
Lawrence, who had offseason back surgery, played the best football of his career in the second half of last season. The coaches credited him with 56 tackles, 31 quarterback pressures and 10 tackles for loss as he became a disruptive force.
In 2014, the Cowboys moved up to the 34th overall pick in a trade with the Washington Redskins to take Lawrence. He did not have a sack as a rookie in the regular season, missing the first eight games of the season with a broken foot.
ESPN's Todd Archer contributed to this report.