The evolutionarily conserved splicing regulator neuro-oncological ventral antigen 1 (NOVA1) plays a key role in neural development and function. NOVA1 also includes a protein-coding difference between the modern human genome and Neanderthal and Denisovan genomes. To investigate the functional importance of an amino acid change in humans, we reintroduced the archaic allele into human induced pluripotent cells using genome editing and then followed their neural development through cortical organoids. This modification promoted slower development and higher surface complexity in cortical organoids with the archaic version of NOVA1 Moreover, levels of synaptic markers and synaptic protein coassociations correlated with altered electrophysiological properties in organoids expressing the archaic variant. Our results suggest that the human-specific substitution in NOVA1, which is exclusive to modern humans since divergence from Neanderthals, may have had functional consequences for our species' evolution.