Harper initially filed for divorce in October 2010, but the couple attempted to reunite several months later.
The reconciliation apparently did not last as the pair were rumoured to have split again in late June.
Dern has now filed paperwork in Los Angeles to reactivate her divorce proceedings, according to TMZ.
The Enlightened actress is reportedly seeking primary physical custody and joint legal custody for their two children, son Ellery Walker and daughter Jaya.
Dern is asking for Harper to pay attorneys' fees as well as spousal and child support payments.
Harper and Dern dated for five years, before marrying on December 23, 2005.
Dern has previously been linked to Hollywood actors Jeff Goldblum, Billy Bob Thornton, Nicolas Cage and her Blue Velvet co-star Kyle MacLachlan.