Endotrophin is a cleavage product of collagen 6 (Col6) in adipose tissue (AT). Previously, we demonstrated that endotrophin serves as a costimulator to trigger fibrosis and inflammation within the unhealthy AT milieu. However, how endotrophin affects lipid storage and breakdown in AT and how different cell types in AT respond to endotrophin stimulation remain unknown. In the current study, by using a doxycycline (Dox)-inducible mouse model, we observed significant upregulation of adipogenic genes in the WAT of endotrophin Tg mice. We further showed that the mice exhibited inhibited lipolysis and accelerated hypertrophy and hyperplasia in WAT. To investigate the effects of endotrophin in vitro, we incubated different cell types from AT with conditioned medium from endotrophin-overexpressing 293T cells. We found that endotrophin activated multiple pathological pathways in different cell types. Particularly, in 3T3-L1 adipocytes, endotrophin triggered a fibrotic program by upregulating collagen genes and promoted abnormal lipid accumulation by downregulating hormone-sensitive lipolysis (HSL) gene and decreasing HSL phosphorylation levels. In macrophages isolated from white AT (WAT), endotrophin stimulated higher expression of the collagen linking enzyme lysyl oxidase (LOX) and M1 pro-inflammatory marker genes. In the stromal vascular fraction (SVF) isolated from WAT, endotrophin induced upregulation of both pro-fibrotic and pro-inflammatory genes. In conclusion, our study provides a new perspective on the effect of endotrophin in abnormal lipid accumulation and a mechanistic insight into the roles played by adipocytes and a variety of other cell types in AT in shaping the unhealthy microenvironment upon endotrophin treatment.