Oil company Exxon Mobil has regained the top slot after Apple shares fell 2.4%, following a 12% drop on Thursday.
Apple, which posted disappointing iPhone sales figures on Wednesday, has seen its shares fall 37% since their record high last September.
Exxon became number one in 2005, traded places with Apple during 2011, and had been number two since early 2012.
At the close on Wall Street, Apple had a market value of $413bn (Â£261bn), against Exxon's of $418bn.
The tech giant has been hit by fears over its future growth, despite record profits.
Although the firm said on Wednesday that it had sold more iPhones (47.8 million) and iPads (22.9 million) in the final three months of last year than in any previous quarter, investors and analysts had expected yet more.
On Thursday, about $50bn was wiped off Apple's value after the biggest daily drop in the firm's stock in four years.
Apple is also facing fierce competition from rivals like Samsung, which accounted for one in four of all mobile phones shipped worldwide last year, according to Strategy Analytics.
Apple's share price rose sharply following a revival under Steve Jobs, who died in 2011, which came about first in computers and then the iPod music player, and was then followed by the iPhone and iPad.
Apple's shares were worth as little as $3.19 in 1997 when it faced the possibility of bankruptcy, and reached a record $702.1 on 19 September.
Apple has lost its crown as the world's most valuable publicly traded company after its shares continued to fall.