A significant number of individuals on combination anti-retroviral therapy (cART) are also chronic alcohol consumers. Alcohol and cART independently induce perturbed intestinal function, but their combined effects on Paneth cells (PCs) and intestinal stem cells (ISCs) remain unclear. Thirty-two adult male Sprague-Dawley rats were divided into 4 groups and treated with normal saline, alcohol treated (AC), cART, and a combination of alcohol and cART (AC+cART) for 90 days.Sections of the small intestine were studied for histomorphology, PC granules, crypts, and ISCs in the jejunum and ileum using hematoxylin and eosin, Alcian Blue Periodic Acid-Schiff, Masson trichrome stains, and immunohistochemistry. This study reveals alcohol-induced collagen increase and cART-induced impairment in the crypts, PC granules, and diminished Musashi-1 expression of ISCs, in the jejunum and ileum. Additionally, depleted goblet cells, crypt depth, and number, but increased intestinal wall thickness and collagen in the ileum of the AC+cART group. Minimal PC granules in the stem cell and transit amplifying zone, with reduced Musashi-1 expression in the jejunum and ileum of the AC+cART group. Moreover, all the independent effects of alcohol and cART are exacerbated in the AC+cART group, resulting in perturbations of the small intestine epithelium, ISC, and PC granules, which may adversely affect the regulation of gut innate immunity, intestinal absorptive function, with adverse health outcomes when exposed to infections. These findings are clinically invaluable in managing patients who receive cART prophylaxis, considering the critical significance of PCs and ISCs in the absorption of medications and necessary nutrients for better treatment outcomes.