Voltage-dependent anion channel (VDAC) is a beta-barrel channel on the mitochondrial outer membrane (MOM) known to transport water-soluble metabolites such as ATP, ADP, and pyruvate. It is central to the regulation of cellular metabolism and serves as a cellular hub given its location at the interface between cytosol and mitochondria. There are three mammalian isoforms of VDAC: VDAC1, 2, and 3. They show high sequence similarity and form similar highly conductive voltage-gated channels in vitro. However, cellular and mice knockout (KO) studies of VDAC isoforms suggest a unique physiological role for each isoform. While VDAC1 KO only leads to mild bioenergetic defects, VDAC3 KO results in male infertility and VDAC2 KO causes developmental impairment in mice. To investigate the role of VDAC isoforms in cellular function, we generated HeLa cells with CRISPR-Cas9 KO of each VDAC isoform. We found isoform-specific differences in mitochondria respiration such as basal and maximal respiration rates for the different isoform KO cells using the Seahorse Metabolic Flux Analyzer. VDAC1 and VDAC3 KO cells showed a significant loss in respiration compared to WT while there was no significant difference detected for VDAC2 KO cells. Interestingly, only VDAC3 KO resulted in complete loss of spare respiratory capacity which could be attributed to defects in the fuel uptake pathway as the VDAC3 KO cells show increased glutamine dependence and loss of flexibility to utilize other metabolites compared to WT HeLa cells. We also detected isoform-dependent differences in calcium signal in mitochondrial intermembrane space (IMS) using IMS targeted calcium sensor (IMS-RGeco1). These results suggest different metabolite and calcium transport functionality for the VDAC isoforms which may contribute to their distinct physiological functions in vivo through isoform-specific regulation of metabolism and calcium signaling.