The goal of this work is to analyze the mitochondrial changes that Rotenone (Rot) treatment can induce in primary cortical neurons that could contribute to the schizophrenia-like phenotype animal model, previously demonstrated by our research group. For this, we previously determined the Rotenone (Rot) concentration in the cortex (1.325nM) from our animal model with a schizophrenia-like phenotype. Then, we treated the primary cortical neurons with Rot 1.325nM for 24h and investigated: the Rot cellular viability treatment by MTT and CytotoxGlo assay; CI enzymatic activity; oxygen consumption rate; and protein levels related to mitochondrial number (TOM40 and TIM23), and mitochondrial fusion (Opa1 and Mfn2) and fission (DRP1). All the results are demonstrated as a percentage of Vehicle (Vehicle=100%;mean±SEM); the statistics were carried out with Students’ t-test. We found that Rot 1.325nM did not reduce the cellular viability (p=0.182;n=7), however, it decreased the CI activity by 15% (n=4). On mitochondrial respiration (n=5) we found a decrease in basal respiration (73.3±9%;p=0.01); non-mitochondrial respiration (84.24±6.48%;p=0.05), ATP-linked respiration (73.1±8.23%;p=0.007), maximum respiratory capacity (76.44±8.3%;p=0.01), and spare capacity (76.3±9.9%;p=0.03). About mitochondrial dynamics (n=6), we saw increased levels of TOM40 (119.3±5%;p=0.05), TIM23 (149.3±11.4%;p=0.006), and DRP1 phosphorylation (129.4±6.9%;p=0.03) without changes in Opa1 (p=0.3) and Mfn2 (p=0.3) levels. Our results demonstrated that 1.325nM of Rot is capable of promoting inhibition of CI activity that led to decreased mitochondrial respiration, increased mitochondrial fission, and possible increase in mitochondrial number. Study supported by FAPESP (2015/02041-1;2021/03021-5;2021/06009-6).