Brucellosis is a common disease in humans and livestock in which treatment and relapse of this disease are one of the significant challenges. This study aimed to evaluate the therapeutic effect of doxycycline-loaded solid lipid nanoparticles (DOX-SLN) on acute and chronic brucellosis and its effect on serum level 25-OH-Vitamin D, C-reactive protein (CRP), and biochemical parameters. The double emulsion method was used for the synthesis of nanoparticles. Serum vitamin D levels, CRP, total protein, albumin, and globulin were measured in rats involved with brucellosis. The therapeutic effect of DOX-SLN was compared with that of free doxycycline.The mean size, Zeta potential, PDI, drug loading, and encapsulation efficiency of the synthesized nanoparticles were 299 ± 34 nm, −28.7 ± 3.2 mV, 0.29 ± 0.027, 11.2 ± 1.3%, and 94.9 ± 3.2%, respectively. Serum vitamin D levels in rats with chronic brucellosis decreased compared to healthy rats. CRP increased in the acute and chronic stages of brucellosis. Serum levels of total protein and albumin were similar in both stages of the disease, but serum globulin levels were increased. According to the results, the serum level of vitamin D was increased in rats after treatment with DOX-SLN. The use of DOX-SLN as an alternative drug for the treatment of chronic brucellosis is recommended.