4-Nonylphenol (4-NP) is a breakdown product of alkylphenolpolyethoxylates and can be found in almost all environmental water matrices. 4-NP can act as environmental stressor on fish, typically causing modulation of hypothalamic–pituitary–interrenal axis (HPI). To examine the effects of the xenoestrogen 4-NP or 17β-estradiol (E2) on induction of stress response mechanisms by evaluating the levels of proopiomelanocortin (POMC)mRNA, heat shock protein 70 (HSP70) mRNA and plasma cortisol, we exposed juvenile sole (Solea solea), under static condition for 7 day, to either 10−6 or 10−8 M4-NP, or 10−8 ME2. In addition, plasma cortisol titers were correlated to the total antioxidant capacity (TAC), one of the oxidative stress parameters. 4-NP treatments resulted in high levels of POMC mRNA, HSP70 mRNA and plasma cortisol. On the contrary, E2 basically down-regulated POMC expression. Moreover, elevated cortisol levels in fish exposed to the highest dose of 4-NP were accompanied by low TAC. These results suggest that 4-NP modulates the sole HPI axis inducing a cortisol-mediated stress response. Specifically, we suggest that 4-NP affects brain POMC mRNA levels via non-estrogen receptor (ER)-mediated mechanism further supporting the ability of 4-NP to target multiple receptor systems.