Objective To investigate the role of circular RNA cleavage and polyadenylation specificity factor 6 (circRNA CPSF6) in the apoptosis of trophoblast cells induced by homocysteine (Hcy) and its mechanism. Methods HTR-8/SVneo human chorionic trophoblast cells were cultured in vitro and divided into control group (0 mmol/L Hcy treatment) and 1 mmol/L Hcy treatment group. Immunofluorescence cytochemical staining was used to detect the expression of caspase-3 in trophoblasts, and Western blot analysis was used to detect the caspase-3 protein level. The mRNA expression level of circRNA CPSF6 was detected by real-time quantitative PCR. Small interfering RNA (siRNA) was used to knock down the expression of circRNA CPSF6 in trophoblast cells. The expressions of caspase-3, caspase-9, Bcl2, and BAX were detected by Western blot analysis. Real-time quantitative PCR was used to detect the expression level of circRNA CPSF6 in the cytoplasm/nucleus of trophoblast cells before and after Hcy treatment. Results Compared with those in the control group, the expressions of caspase-3 and circRNA CPSF6 mRNA in the Hcy treatment group significantly increased. After knocking down circRNA CPSF6, the expression of caspase-3 decreased, and the mitochondrial apoptosis pathway was inhibited. In normal cultured trophoblast cells, circRNA CPSF6 was expressed in large amounts in the cytoplasm, and after Hcy treatment, circRNA CPSF6 was mainly expressed in the nucleus. Conclusion The mitochondrial apoptotic pathway is activated by circRNA CPSF6 nuclear translocation to promote trophoblast apoptosis induced by Hcy.