ABSTRACT During adipogenesis, preadipocytes' cytoskeleton reorganizes in parallel with lipid accumulation. Failure to do so may impact the ability of adipose tissue (AT) to shift between lipid storage and mobilization. Here, we identify cytoskeletal transgelin 2 (TAGLN2) as a protein expressed in AT and associated with obesity and inflammation, being normalized upon weight loss. TAGLN2 was primarily found in the adipose stromovascular cell fraction, but inflammation, TGF‐β, and estradiol also prompted increased expression in human adipocytes. Tagln2 knockdown revealed a key functional role, being required for proliferation and differentiation of fat cells, whereas transgenic mice overexpressing Tagln 2 using the adipocyte protein 2 promoter disclosed remarkable sex‐dependent variations, in which females displayed “healthy” obesity and hypertrophied adipocytes but preserved insulin sensitivity, and males exhibited physiologic changes suggestive of defective AT expandability, including increased number of small adipocytes, activation of immune cells, mitochondrial dysfunction, and impaired metabolism together with decreased insulin sensitivity. The metabolic relevance and sexual dimorphism of TAGLN2 was also outlined by genetic variants that may modulate its expression and are associated with obesity and the risk of ischemic heart disease in men. Collectively, current findings highlight the contribution of cytoskeletal TAGLN2 to the obese phenotype in a gender‐dependent manner.—Ortega, F. J., Moreno‐Navarrete, J. M., Mercader, J. M., Gómez‐Serrano, M., García‐Santos, E., Latorre, J., Lluch, A., Sabater, M., Caballano‐Infantes, E., Guzmán, R., Macías‐González, M., Buxo, M., Gironés, J., Vilallonga, R., Naon, D., Botas, P., Delgado, E., Corella, D., Burcelin, R., Frühbeck, G., Ricart, W., Simó, R., Castrillon‐Rodríguez, I., Tinahones, F. J., Bosch, F., Vidal‐Puig, A., Malagón, M. M., Peral, B., Zorzano, A., Fernández‐Real, J. M. Cytoskeletal transgelin 2 contributes to gender‐dependent adipose tissue expandability and immune function. FASEB J. 33, 9656–9671 (2019). www.fasebj.org