Accumulating evidence determined that lncRNAs play multiple roles in cell progression in colorectal cancer (CRC). Long noncoding RNA (lncRNA) hepatocyte nuclear factor 1 homeobox A (HNF1A)-antisense RNA 1 (AS1) has been identified to affect cell growth and disease diagnosis in various cancers, including CRC. However, the underlying regulatory mechanism of HNF1A-AS1 in cell progression and glycolysis has not been fully explored in CRC.The expression of HNF1A-AS1, microRNA-124 (miR-124) and Myosins of class VI (MYO6) was detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The analysis of glucose consumption, lactate production and hexokinase 2 (HK2) protein level was used to assess glycolysis in cells. The protein level of HK2 and MYO6 was measured with Western blot. Cell migration and invasion were evaluated using the transwell assay. The relationship among HNF1A-AS1, miR-124 and MYO6 was determined via luciferase reporter and RNA immunoprecipitation (RIP) assay.In this study, we found that HNF1A-AS1 was upregulated in CRC tissues and cell lines. Functional experiments determined that reduction of HNF1A-AS1 or promotion of miR-124 inhibited cell migration and invasion as well as glycolysis in CRC cells. What' more, luciferase reporter assay manifested that miR-124 was a target of HNF1A-AS1 and MYO6 was a target mRNA of miR-124 in CRC cells. Additionally, reverse experiments showed that the effects of si-HNF1A-AS1 on colorectal cancer cells were impaired by anti-miR-124 and the effects of high miR-124 expression on CRC cells were rescued by upregulating MYO6. HNF1A-AS1 regulated MYO6 expression via targeting miR-124 in CRC cells.In this study, we first found that HNF1A-AS1 regulated cell migration, invasion and glycolysis via modulating miR-124/MYO6 in CRC cells.