Vitamin K has important physiological functions which relate to blood coagulation (its classical role), bone health, inhibition of arterial calcification, as well as anti-inflammatory effects. National nutrition survey data have shown that over half of all adults in Ireland and the UK have low vitamin K1 intakes (£1 μg/kg body weight/d). Vitamin K biofortification of food may be an important complementary food-based approach for improving vitamin K intakes. Our study aimed to explore the feasibility of producing vitamin K-biofortified eggs via increasing the vitamin K3 content of the hen diet, and to examine any effects on hen performance and egg/eggshell quality parameters. A 12-week hen feeding trial was conducted in the Agri-Food and Biosciences Institute, Belfast, UK. Hyline chickens (n = 128) were randomized into 4 treatment (T) groups (n = 32 hens/group) and fed diets containing 3 (T1-industry standard), 12.9 (T2), 23.7 (T3) and 45.7 (T4) mg vitamin K3/kg of feed. Hens were provided feed ad libitum and feed intake was recorded weekly. Eggs were collected daily and weighed. While egg quality and vitamin K content was assessed at week 0, 4, 6, 8 and 12, the trial endpoint (week 12) data was prioritized for the current analysis. Total vitamin K content (i.e., vitamin K1 plus menaquinone (MK)-4 and MK-7) of composite samples (n = 12 eggs/treatment), measured using a novel, sensitive liquid chromatography-mass spectrometry method at Teagasc, increased from 22.4 μg/100 g (whole egg) in T1 (control and commercial level of vitamin K3) to 57.8 μg/100 g in T4. MK-4 was the most abundant form of vitamin K found in the eggs. Eggshell weight and eggshell thickness in T2 and T3 (but not T4) were significantly higher than in T1 (by 20–28%, P < 0.05). Likewise, based on colourimetric assessment, yellowness (parameter b*) of the egg yolk in all three biofortified groups was significantly higher compared to T1 (by 27–45%, P < 0.05). Haugh unit of the eggs and hen performance parameters, such as feed intake, feed conversion ratio and egg production, were unaffected by vitamin K-biofortification. In conclusion, increasing the level of addition of vitamin K3 to hen feed significantly increased the total vitamin K content of eggs, and without any evidence of negative effects on egg quality or hen performance. Consumption of an average sized (60 g) vitamin K-biofortified egg could contribute an additional 35 μg total vitamin K. The effects on eggshell parameters could be of importance in terms of revenue loss due to breakages.