The stroma plays a fundamental role in the function of different glandular systems. In the prostate, the stroma is responsible for the development and maintenance of the differentiated state of the epithelium. Nicotine induces tobacco dependence and promotes morphological alterations in the epithelial compartment. However, its effects on the prostate stroma are unclear. The objective of this study was to evaluate the morphology of the stromal microenvironment in the ventral prostate lobe of rats submitted to chronic nicotine administration. Twenty rats (Rattus norvegicus) were divided into two groups: 10 animals received subcutaneous nicotine and 10 animals received physiological saline by the same route. After treatment, samples were collected from the ventral prostate lobe, processed and submitted to histology, histochemistry, and ultrastructural analysis by transmission and scanning electron microscopy. The level of circulating testosterone was also analyzed. The results showed a significant increase in the density of type I collagen (56.3% to 85.9%, P < 0.01) and a decrease in the density of type III collagen (43.7% to 14.1%, P < 0.01). In addition, there was a qualitative increase in elastic fibers and in the number of smooth muscle cells with a secretory phenotype. Circulating testosterone levels were significantly reduced (898.3 to 363.1 ng/mL, P < 0.01). The results showed that nicotine modifies different components of the prostate stroma, suggesting that this drug is a risk factor for morphofunctional alterations in the prostate gland. Microsc. Res. Tech. 2012. © 2011 Wiley Periodicals, Inc.