Police believe the truck was travelling in front of the bus when its trailers dislodged and blocked the highway in both directions, causing the crash and trapping the driver, who died.

The accident happened near Pimpinio about 2am on Thursday, with at least nine people injured, Ambulance Victoria said.

Paramedics treated nine people for various injuries, and two were flown by air ambulance plane to Melbourne.

A man in his 40s with leg injuries was flown to Royal Melbourne Hospital in a stable condition and a woman in her 60s with arm injuries was flown to The Alfred hospital, also in a stable condition.

Another seven people aged from their teens to those in their 70s were taken to Wimmera Health Care in Horsham after being treated by paramedics.

More than 30 other bus passengers will also be assessed at Wimmera Health Care.

The Western Highway is closed in both directions following the crash, with diversions in place and detectives at the scene.

Wimmera Health Care Group chief executive Catherine Morley said ambulances and the health care group's bus transported more than 40 patients due to be treated in Horsham.

"Obviously, an event like this and number of people needing to come into our Accident and Emergency Department is unprecedented. In saying that, we have been extremely fortunate that the people who have been brought in have relatively minor injuries and are stable," Ms Morley said.

"It has, however, obviously been a very traumatic experience for everyone involved and we are caring for patients on all fronts."

She said extra nurses, doctors and support staff were called in to ensure those injured were looked after.

"This has been a huge team effort to ensure everyone is cared for in the best way possible. We want to thank everyone involved from the huge ambulance crew which involved every resource this side of Ararat, police and SES members on site at the scene; to the extra staff who were called in during the middle of the night to make sure we had enough people not just to medically treat people, but to transport people, feed people and make sure they are comfortable," Ms Morley said.

An emergency management centre has been set up.

"We have teamed up with Horsham Rural City Council and have set up an emergency management centre to help cater for people once they have been medically cleared," Ms Morley said.

"We are working with the cleared patients to call their families, make transport arrangements and keep them calm, warm and fed.

"We would appreciate if the privacy of the patients could be respected at this time. Patient care is our priority and all our energy is being directed into ensuring we are looking after people the best we can."

Detectives from the Major Collision Investigation Unit are attending the scene.

Anyone who witnessed the incident or with dash cam footage is urged to contact Crime Stoppers on 1800 333 000 or submit a confidential report online at www.crimestoppersvic.com.au

The crash comes on the morning after Ballarat hosted the Towards Zero Regional Road Safety Forum, with a focus on the sky-rocketing road toll in the state.