Serum concentrations of 25-hydroxycholecalciferol [25(OH)D3] in broiler embryos at 19 d of incubation:doi:have been shown to increase 3 times by the in ovo injection of 0.60 μg 25(OH)D3 on 18 doi. In this trial, effects of the injection of 25(OH)D3 at 18 doi on the yolks and sera of male and female Ross×Ross 708 broiler embryos were assessed. On 18 doi, embryonated eggs that were set in a single stage incubator were assigned to 6 replicate trays within each of 2 injection treatment groups. Treatments included those injected with 100 μL commercial diluent (control) and those injected with 0.60 μg 25(OH)D3 in 100 μL commercial diluent. On 19 doi, embryos and their yolk sacs were extracted for determination of sex, yolk weight, BW, serum CA and phosphorous concentrations, and yolk CA, phosphorous, moisture, dry matter, and lipid concentrations. The weight of female embryos with their attached yolk sacs as a percentage of set egg weight was greater than that of males (P=0.03). There were treatment×sex interactions for the weight of embryos with their attached yolk sacs relative to 19 doi egg weight (P=0.05) and for yolk CA concentration (P=0.004). In eggs that received 25(OH)D3, the weight of female embryos with their attached yolk sacs relative to 19 doi egg weight was higher than that of males (P=0.005), and percentage yolk CA was higher in control eggs containing female embryos in comparison to those containing males (P=0.007). An injection of 0.60 μg 25(OH)D3 at 18 doi eliminated yolk CA differences associated with embryo sex, suggesting that 25(OH)D3 may influence sex-related differences in the rate of yolk CA absorption by broiler embryos and may be related to subsequent sex-related differences in posthatch bone strength.