Background: Long non-coding RNA (lncRNA) hypoxia inducible factor 1α-antisense RNA 1 (HIF1A-AS1) serves critical roles in cardiovascular diseases (CVDs). Vascular endothelial cells (VECs) are vulnerable to stimuli. Our previous study revealed that knockdown of HIF1A-AS1 reduces palmitic acid-induced apoptosis and promotes the proliferation of human VECs (HUVECs); however, the underlying mechanism remains unclear. Material and Methods: Cell Counting Kit-8, flow cytometry, transwell invasion, and wound healing were applied to detect the function of HUVECs. Moreover, miRNA sequencing (miRNA-seq) and RNA sequencing (RNA-seq) were conducted to uncover its underlying mechanism. Quantitative Polymerase Chain Reaction (qPCR) was implemented to assess the accuracy of miRNA-seq. A co-expression network was generated to determine the relationship between differentially expressed miRNAs (DEmiRNAs) and differentially expressed genes (DEGs). Results: Knockdown of HIF1A-AS1 promoted the proliferation, migration, and invasion but reduced the apoptosis of HUVECs, and the overexpression of this lncRNA had the opposite effect. Numerous DEmiR-NAs and DEGs were identified, which might contribute to this phenomenon. Multiple target genes of DEmiRNAs were associated with cell proliferation and apoptosis, and overlapped with DEGs identified from RNA-seq. Finally, the network manifested that lncRNA HIF1A-AS1 moderated the function of HUVECs by not only regulating the expression of some genes directly but also by influencing a few miRNAs to indirectly mediate the expression of mRNAs. Conclusions: The results suggested that HIF1A-AS1 might regulate HU-VEC function by not only regulating the expression of some genes directly but also by influencing some miRNAs to indirectly mediate the expression level of mRNA.