The COVID-19 pandemic has claimed over 6.5 million lives worldwide and continues to have lasting impacts on the world's healthcare and economic systems. Several approved and emergency authorized therapeutics that inhibit early stages of the virus replication cycle have been developed however, effective late-stage therapeutical targets have yet to be identified. To that end, our lab identified that 2',3' cyclic-nucleotide 3'-phosphodiesterase (CNP) inhibits SARS-CoV-2 virion assembly. We show that CNP inhibits the generation of new SARS-CoV-2 virions, reducing intracellular titers without inhibiting viral structural protein translation. Additionally, we show that targeting of CNP to mitochondria is necessary for inhibition, blocking mitochondrial depolarization and implicating CNP's proposed role as an inhibitor of the mitochondrial permeabilization transition pore (mPTP) as the mechanism of virion assembly inhibition. We also demonstrate that an adenovirus expressing virus expressing both human ACE2 and CNP inhibits SARS-CoV-2 titers to undetectable levels in lungs of mice. Collectively, this work shows the potential of CNP to be a new SARS-CoV-2 antiviral target.