This study aimed to evaluate the effects of dietary boric acid with vitamin D₃ on laying performance, eggshell quality, and mineral content and fatty acid composition of egg yolk in hens. In the experiment, 160 late-phase laying hens were equally divided into four groups, 40 birds each (10 replicates of 4 hens each). The study included a control group and three treatment groups. In the control group, there was no additive, while the experimental groups contained: 1) vitamin D₃ 3000 IU/kg + 120 ppm boric acid, HDB; 2) vitamin D₃ 300 IU/kg + 120 ppm boric acid, LDB; and 3) 120 ppm boric acid, B; respectively. After 12 weeks of feeding, the egg mass was significantly reduced in HDB group in comparison to the control and feed efficiency was increased in HDB group in comparison to the control and B groups (P < 0.05). No significant differences were observed in eggshell quality among groups (P > 0.05). Egg yolk cholesterol content was decreased in LDB group in comparison to B group (P < 0.05). The concentration of C18:3 fatty acid was the highest (P < 0.05) in B group. In comparison to the control group, the C16:0 fatty acid concentration was lower in B group (P < 0.05). So, the combination of boric acid (120 ppm) and a high dose of vitamin D3 (3000 IU/kg) is not recommended in laying hen feeding as it negatively influences laying performance parameters. On the other hand, although the supplementation of 120 ppm boric acid without additional vitamin D3 increases egg yolk omega-3 fatty acid content, the cholesterol-reducing effect (but without fatty acid profile change) may be obtained only when boric acid is added simultaneously with a low level of vitamin D₃ (300 IU/kg), which can be used to produce functional food.