The motorway is closed westbound between junctions 4 (Darwen / Blackburn South) and 3 (Blackburn West / Wheelton / Walton) after the crash at around 5.30am.
Police, fire and ambulance crews are working at the scene and motorists are advised to avoid the area until further notice.
All traffic was held on both sides of the motorway after the crash, but the eastbound carriageway has since been released.
Traffic on the westbound is congested back to junction 5 (Guide / Shadsworth) and diversions are now in place (see route below).
Routes around Feniscowles are also busy with traffic trying to avoids the closure.
North West Ambulance Service said three vehicles were involved and two casualties have been taken to hospital.
A spokesman said: “We’ve responded to a serious RTC on the M65 following a call at 5.24am this morning.
“Three response vehicles and two ambulances attended the scene. Two casualties have been taken to hospital.”
A spokesman for Lancashire Police added: “We were called at 5.27am to reports of a serious collision on the westbound carriageway.
“Road closures are currently in place between J4 and J3.
"Where possible, we would advise avoiding the area and using alternative routes.
“Emergency services remain at the scene. We will provide an update when we have more information.”
National Highways added: "Drivers heading along the M65 in Lancashire are being advised the westbound carriageway is currently closed between junction 4 and junction 3 south of Blackburn and is likely to remain closed beyond this morning’s peak travel period.
"The closure was put in place following a serious collision, involving two vehicles, shortly before 6am this morning.
"A police investigation is underway and this is likely to take several hours.
"A diversion between junction 4 and junction 3 is in place with drivers being asked to follow the ‘hollow square’ symbol – using the northbound A666, westbound A6062 from Ewood Park across to the southbound A674 which drivers should follow to reach and re-join the motorway at junction 3.
"Congestion on the diversion route is already very heavy so drivers should delay journeys or seek alternative routes away from this section of the M65."
Road users are advised to follow the Hollow Square diversion symbol on road signs
- Exit the M65 westbound at J4. At the M65 J4 roundabout, follow Earcroft Way.
- At Earcroft Way/A666 junction, turn right and follow the A666 northbound Bolton Road.
- At the A666/A6062 junction, turn left and follow the A6062 westbound Livesey Branch Road.
- At the A6062/A674 junction, turn left and follow the A674 westbound Moulden Road.
- At the A674/A6061 junction, turn left and follow A674 southbound Finnington Road.
- At the A674 M65 J3 roundabout, re-join the M65 at J3.