Autophagy is a complex degradation pathway through which damaged or dysfunctional proteins and organelles are removed. Its pharmacological modulators have been extensively used in a wide range of basic research and preclinical studies. However, the effects of these inhibitors on metabolism, in addition to autophagy inhibition, are not fully elucidated. Chloroquine is a clinically relevant compound that inhibits autophagy by preventing the fusion of autophagosomes with lysosomes. In this study, we aimed to examine the effect of chloroquine on mitochondrial quality control and respiratory function by utilizing 3T3-L1 mouse adipocytes treated with chloroquine at various time points. We found that chloroquine could disturb genes related to mitochondrial fission, biogenesis, and mitophagy, leading to mitochondrial DNA damage. Although the inhibition of autophagy by chloroquine resulted in an increased prohibitin expression, respiratory function was downregulated in a time-dependent manner. Moreover, chloroquine treatment induced oxidative stress, apoptosis, and metabolic dysregulation. These data demonstrated that chloroquine significantly affected mitochondrial respiratory function and metabolism, which was consistent with impaired mitochondrial quality associated with autophagy inhibition.