Background and Aims Studies have shown that changes in the metabolism of pyruvate play an important role in the occurrence and development of colorectal cancer, and abnormal expression of microRNAs (miRNAs) in colorectal cancer stem cells may be closely related to pyruvate metabolism. The author's previous analysis of the TCGA database found that miR-520c-3p was upregulated in colorectal cancer and correlated with prognosis. However, it is not clear whether miR-520c-3p is involved in pyruvate metabolism. Therefore, this study explores the relationship between the expression of miR-520c-3p in colorectal cancer stem cells and pyruvate metabolism.Methods Human colon cancer cell lines were selected, and colorectal cancer stem cells were isolated and purified from them. Changes in the proliferation ability, pyruvate oxidation levels, and lactate production in colorectal cancer stem cells and colorectal cancer cells were detected after overexpression or knockdown of miR-520c-3p. Cells were incubated with _D-(U-¹³C) glucose, and mass isotopomer analysis was used to trace the fate of glucose-derived carbons. MiR-520c-3p functional substrates were analyzed and identified through miRNA sequence analysis and genetic approaches.Results After overexpression of miR-520c-3p, the proliferation ability of colorectal cancer stem cells significantly increased, while the levels of pyruvate oxidation significantly decreased and lactate production significantly increased (all P<0.05). When cultured with _D-(U-¹³C) glucose, the unlabeled citrate (m+0) in colorectal cancer stem cells significantly increased, while the higher-order citrate isotopomers (m+1, m+4, and m+5) significantly decreased (all P<0.05). Conversely, after knockdown of miR-520c-3p, the above-mentioned changes in colorectal cancer stem cells were reversed (all P<0.05). Overexpression or knockdown of miR-520c-3p had no significant effect on the above-mentioned indicators in colorectal cancer cells (all P>0.05). MiR-520c-3p could target the 3'UTR of mitochondrial pyruvate carrier 1 (MPC1) mRNA (P<0.05). After overexpression of miR-520c-3p, both mRNA and protein levels of MPC1 in colorectal cancer stem cells significantly decreased, while the opposite was observed after knockdown of miR-520c-3p (all P<0.05). Analysis of TCGA database showed that colorectal cancer patients with low MPC1 expression had poorer prognosis (P<0.05). Knockdown of MPC1 led to a significant decrease in pyruvate oxidation levels, significant increase in lactate production, and significant enhancement of proliferation ability in colorectal cancer stem cells (all P<0.05). When cultured with _D-(U-¹³C) glucose, the unlabeled citrate (m+0) significantly increased, while the higher-order citrate isotopomers (m+1, m+4, and m+5) significantly decreased in colorectal cancer stem cells with MPC1 knockdown (all P<0.05). Meanwhile, after knockdown of both miR-520c-3p and MPC1, there were no significant changes in pyruvate oxidation levels, lactate production, and proliferation ability in colorectal cancer stem cells (all P>0.05).Conclusions The high expression of miR-520c-3p in colorectal cancer is associated with poor prognosis, and its mechanism may be related to its regulation of pyruvate metabolism in colorectal cancer stem cells through targeting MPC1, promoting the proliferation of colorectal cancer stem cells.