Its profits "are available to support the full range of the IRGC's illicit activities, including WMD proliferation and support for terrorism", the department said.
Khatam al-Anbiya was first hit with US sanctions by the administration of George Bush, the former president, in 2007.
Wednesday's penalties apply to Qasemi and Khatam al-Anbiya subsidiaries, the Fater Engineering Institute, the Imensazen Consultant Engineers Institute, the Makin Institute and the Rahab Institute.
Stuart Levey, the US treasury undersecretary for terrorism and financial intelligence, said: "Today's action exposing Khatam al-Anbiya subsidiaries will help firms worldwide avoid business that ultimately benefits the IRGC and its dangerous activities."
The announcement comes as Washington tries to rally international support against Tehran's nuclear programme, seeking fresh sanctionsby the UN Security Council, which has already hit Iran with three sets of sanctions.
Speaking at a White House press briefing on Tuesday, Barack Obama, the US president, said the international community was pursuing "a significant regime of sanctions" against what Washington called Iran's intransigence.
Obama's comments came after Tehran said on Tuesday that it had begun the process of enriching uranium at higher levels, fuelling fears that the process could eventually be used to produce nuclear weapons.
It had been reported in Iranian state media that the process of refining uranium to 20 per cent purity had begun earlier in the week, but Tehran told nuclear inspectors on Wednesday that it had only been making preparations for the higher enrichment.
"We were told that it was expected the facility would begin to produce up to 20 per cent [uranium] within a few days," an internal memo by the UN nuclear watchdog, the International Atomic Energy Agency (IAEA), said.
The IAEA memo indicated that, for now at least, the Iranian operation was modest in scale.
The memo from Yukiya Amano, the IAEA chief, said Iran had recalibrated 164 centrifuges, out of its 8,000 enrichment machines, for higher-scale enrichment at its Natanz plant.
He said Iran notified the IAEA of the plan on Monday and the IAEA quickly asked Iran not to launch it before inspectors could adjust their monitoring procedures, as well as get clarification on the expected duration of the new programme and technical details.
"On Wednesday, when agency inspectors arrived at the pilot plant, they were informed that Iran had begun to feed the low-enriched uranium into one cascade the previous evening for [test] purposes," the memo said.
A cascade is 164 centrifuges hooked up in series that spin and respin uranium gas to the required enrichment level.
The document, relying on onsite reports from IAEA inspectors, also cited Iranian experts at the enrichment site at Natanz as saying that only about 10kg of low enriched uranium out of 1.8 tonnes, had been fed into the cascade for further enrichment.
A nuclear bomb requires uranium of about 90 per cent purity, but nuclear experts say getting to 20 per cent is a big step because low-level enrichment is the most time-consuming and difficult stage of the process.