The television commercial calls Casey the deciding vote for President Barack Obama's health care overhaul law, although he was one of 60 who backed the measure. It also criticizes the first-term senator on energy.
"Sen. Casey. Washington has changed him, and he's let us down," says the ad.
Rob Engstrom, national political director for the chamber, declined to discuss the amount of the ad buy. The chamber's move comes just days after the National Republican Senatorial Committee invested $500,000 in the race as polls show a closer contest between Casey and Republican Tom Smith, a wealthy tea party candidate who has spent more than $16 million on his bid.
"Pennsylvania absolutely is in play," Engstrom said.
The GOP investment in the Senate race also comes as Republican presidential candidate Mitt Romney made plans to hold a rally in the state on Sunday.
Democrats insist that Casey has the advantage in the state and argue that polls showed him with a lead.
"The late timing of the buy is curious given how saturated the airwaves have become," said Larry Smar, Casey's campaign manager. "It isn't, however, new that Tom Smith is being backed by Washington, D.C., special interests and Tea Party groups."
Smar said Casey has a record of promoting energy jobs.
Casey is bidding for a second term against a Republican who didn't have the backing of the state's Republican governor but scored a surprise win in the primary. A political newcomer, Smith made a fortune in coal mining and has been on course to outspend Casey by 2-to-1 since July 1.
Last week, the Majority PAC, a Democratic political action committee, began running TV ads in Pittsburgh to help Casey. The commercial criticizes Smith for his plans to eliminate the federal Education Department, arguing that it would mean less money in classrooms and less aid for college students.
The Smith campaign said he has not ruled out keeping some Education Department functions, such as Pell grants.
Casey, a moderate Democrat, is the son of a former governor, a veteran of several statewide races and popular with labor unions in a strong labor state. Although Republicans are investing in the race, strong Democratic turnout in Philadelphia, especially in a presidential election year, often dashes GOP hopes.