Abstract: Objective To investigate the effect of remimazolam combined with sevoflurane on the malignant biological behavior of colorectal cancer cells and its underlying mechanisms. Methods Human colorectal cancer cell line HCT116 was used as the research subject in the study. Cells were divided into the following groups: a control group, a sevoflurane (Sev) group, a remimazolam (Rem) group, a combined treatment group, a Midivi-1 (mitochondrial autophagy inhibitor) group, a combined treatment + Midivi-1 group, a combined treatment + oe-HIF-1α group, and a combined treatment + si-HIF-1α group. The MTT assay, scratch assay, and Transwell assay were used to evaluate cell proliferation, migration, and invasion. Flow cytometry was used to detect cell apoptosis. JC-1 staining was used to assess mitochondrial membrane potential. MitoSOX Red staining was used to detect mitochondrial ROS generation. Transmission electron microscopy was used to observe mitochondrial autophagosome. Immunofluorescence was used to detect Mito-Tracker/LC3 co-localization. Western blot was employed to detect the expression of mitochondrial autophagy and HIF-1α/BNIP3 pathway-related proteins. Results Compared with the control group, the Sev group, Rem group, and combined treatment group exhibited significant reduction in cell survival rate, wound healing rate and invasion rate, increases in the apoptosis rate, the levels of Bax and cleaved caspase-3 proteins, decreases in Bcl-2 protein expression and the JC-1 aggregate/monomer fluorescence ratio, increases in mitochondrial ROS content and Mito-Tracker/LC3 colocalization fluorescence intensity, enhancement in mitochondrial autophagy body formation, decreased p62 protein expression, and increased levels of LC3Ⅱ/Ⅰ, PINK1, Parkin, HIF-1α, and BNIP3 proteins. The combined treatment group showed the most significant effect (P<0.05). Compared with the combined treatment group, the combined treatment + Midivi-1 group showed increases in cell survival rate, wound healing rate and invasion rate, decreases in the apoptosis rate, and weakened Mito-Tracker/LC3 colocalization fluorescence intensity. The combined treatment + oe-HIF-1α group showed elevated expression of HIF-1α and BNIP3 proteins and enhanced Mito-Tracker/LC3 colocalization fluorescence intensity. The combined treatment + si-HIF-1α group exhibited decreased expression of HIF-1α and BNIP3 proteins, with a reduced Mito-Tracker/LC3 colocalization fluorescence intensity. All differences were statistically significant (P<0.05). Conclusions Remimazolam combined with sevoflurane may inhibit the proliferation, migration, and invasion of colorectal cancer cells, and promote apoptosis, by activating mitochondrial autophagy mediated by the HIF-1α/BNIP3 pathway.