Di-n-butyl phthalate (DBP) is an endocrine-disrupting chemical (EDC) widely used in the production of plastics, synthetic materials, and cosmetics; it is therefore widespread in the environment as a contaminant. In recent years, DBP has been increasingly associated with metabolic and reproductive dysfunctions. Considering the growing evidence linking metabolic syndrome (MetS) to male infertility, this study explores the effects of DBP on prostate physiology and adipocyte differentiation, which are considered interconnected biological pathways. Using the non-tumor human prostate cell line PNT1A, DBP was analyzed individually and in combination with endogenous hormones: testosterone (T) and 17β-oestradiol (E2), to simulate environmental exposure conditions. The results showed that DBP and all tested mixtures increased cell viability by activating both the oestrogen receptor (ERα) and the androgen receptor (AR). DBP modulated the expression of steroid receptors in a non-monotonic, hormone-specific manner, inducing delayed activation of ERα and AR compared to endogenous hormones. This delay could lead to the activation of proliferation and migration pathways at inappropriate times, altering prostate physiology and increasing susceptibility to neoplastic transformation. Given the central role of ERα, the effects of DBP were subsequently analyzed on adipogenesis in 3T3-L1 preadipocytes. Abnormal activation of ERα led to aberrant regulation of adipogenic genes PPAR-γ and C/EBPα, resulting in increased lipid accumulation (Oil Red O) and overexpression of adipogenic markers. Overall, DBP emerges as an endocrine-disrupting chemical capable of profoundly altering prostate and adipose physiology, suggesting a potential mechanistic link between environmental exposure, metabolic dysfunction, and male infertility.