Human mitochondrial transcription factor A (TFAM) has been implicated in promoting tumor growth and invasion. TFAM activates mitochondrial DNA (mtDNA) transcription, and affects nuclear gene expression through mitochondrial retrograde signaling. In this study, we investigated the effects of TFAM depletion on the morphology and transcriptome of MKN45 gastric cancer cells. Morphology alteration became visible at 12 h after TFAM knockdown: the proportion of growth-arrested polygonal cells versus oval-shaped cells increased, reaching a half-maximum at 24 h and a near-maximum at 36 h. TFAM knockdown upregulated four genes and downregulated six genes by more than threefold at 24 h and similarly at 48 h. Among them, the knockdown of CFAP65 (cilia and flagella associated protein 65) or PCK1 (cytoplasmic phosphoenolpyruvate carboxykinase) rescued the effects of TFAM depletion on cell morphology and proliferation. PCK1 was found to act downstream of CFAP65 in calcium-mediated retrograde signaling. Furthermore, mtDNA depletion by 2′,3′-dideoxycytidine was sufficient for induction of CFAP65 and PCK1 expression and inhibition of cell proliferation, but oxidative phosphorylation blockade or mitochondrial membrane potential depolarization was not. Thus, the TFAM–mtDNA–calcium–CFAP65–PCK1 axis participates in mitochondrial retrograde signaling, affecting tumor cell differentiation and proliferation.