Increased fat cell size, feature of obesity as well as of subjects with T2D familiarity (FDR), results in adipose tissue (AT) hypertrophy. This condition is due to inability to recruit new adipocytes through adipogenesis of resident preadipocytes (preAD), that causes AT dysfunction and insulin resistance (IR). Thus, identifying new adipogenesis regulators may lead to novel therapies for treating AT hypertrophy. We demonstrated that the HOXA5 gene is required for human adipogenesis. However, the underlying mechanism remains elusive. We aim to understand the transcriptional pathways regulated by HOXA5 during preAD differentiation. By RNA-Seq, we examined the transcriptomic effects of HOXA5 silencing on human preAd. Pscan and Chip-Seq data were used to find the HOXA5 motif in the promoters of the differentially expressed genes (DEG). Relevant findings were validated in preAd from an FDR cohort and in AT of obese subjects. RNA-Seq revealed 1401 DEG in HOXA5-silenced preAd vs control cells (FC≥2; padj≤0.05). Of these, 524 genes were upregulated, while 877 were downregulated upon HOXA5 silencing. Pathway analysis showed that HOXA5 loss upregulated genes inhibiting adipogenesis, including pro-inflammatory cytokine- and WNT-pathways, while downregulated genes related to fat cell differentiation (PPARγ-signaling), which is in line with the role of HOXA5 in promoting adipogenesis. Using public HOXA5 ChIP-Seq data and the Pscan tool, we identified direct HOXA5 targets among the DEG. HOXA5 recognizes genes in three broad categories: adipocyte biology, AT remodelling and uncharted role in adipogenesis. To validate this finding, ADH1B and ASPA genes were selected and analyzed by qPCR. The results confirmed their downregulation in preAd lacking HOXA5 (p<0.001), supporting a direct regulation by HOXA5. In preAd from FDR, ADH1B and ASPA expression was reduced and significantly correlated with HOXA5 mRNA levels. ADH1B and ASPA expression, in FDR subjects, negatively correlated with AT hypertrophy and triglyceride levels (p<0.05) while positively associated with insulin sensitivity (euglycemic clamp; p<0.05). Intriguingly, in AT of obese subjects, we showed that ADH1B and ASPA expression positively correlated with HOXA5 expression and negatively correlated with BMI, triglyceride levels and IR (HOMA-IR; p<0.05). HOXA5 is a crucial regulator of human adipogenesis, controlling a previously unknown set of metabolically relevant genes whose dysregulation may cause AT hypertrophy.