Primary mitochondrial diseases are caused by mutations in genes required for mitochondrial function. The primary mitochondrial disease complex I deficiency results from loss-of-function mutations in subunits of mitochondrial respiratory complex 1 and is the most common cause of childhood mitochondrial disease. Complex I deficiency causes severe neurological deficits in patients, but we have little understanding of how individual subtypes of neurons are affected and the underlying mechanisms.We used knockdown of the complex I subunit ND-75 in Drosophila (the homolog of NDUFS1) to investigate the consequences of complex I deficiency in individual neuronal subtypes. ND-75 knockdown (ND-75 KD) in dopaminergic neurons caused changes in locomotion, appetite, sleep and circadian rhythm. By contrast, ND-75 KD in serotonergic neurons only mildly affected circadian rhythm, suggesting that neuronal subtypes respond differently to complex I deficiency. To investigate this in depth, we performed single nuclear RNA-sequencing (snRNA-seq) of the whole brain from flies with pan-neuronal ND-75 KD. The number of differentially regulated genes (DEGs) in dopaminergic ND-75 KD neurons was 10-fold higher than the number of DEGs in serotonergic ND-75 KD neurons, mirroring the phenotypic differences. Moreover, gene ontology analysis showed striking differences in the functional classes of DEGs in dopaminergic and serotonergic ND-75 KD neurons. The snRNA-seq data also revealed a novel class of neurons in the ND-75 KD brain expressing the stress response factor ATF4.Our data provide new insight into the transcriptional, functional and stress responses to complex I deficiency in single neurons.