Abstract: Environmental parameters generally determine the incubation performance and chick quality, one of which is carbon dioxide (CO2). However, the mechanism still remains unclear, particularly the effects of high CO2 levels during early incubation on the hatching quality. In this study, three batches of fertile eggs from Jinghong No. 1 were incubated in two small-scale incubators, in order to investigate the influence of normal and higher CO2 levels during the early stage of incubation (0-10 d). The treatment and control incubator maintained the CO2 concentration of 1% and below 0.25%, respectively. The incubators were also swapped for the next batch. A higher CO2 level was controlled during the incubation using a purpose-built system with CO2 sensors and a CO2 gas adding unit. The specific parameters were measured, including hatchability, chick quality, embryo weight, and relative embryo weight at the 6th, 9th, 12th, 15th, and 18th day, vascular development density of allantoic chorion (CAM) at day 11, albumen pH at day 0, day 3, day 6, day 9, day 12 and day 15, and the calcium content of eggshells and embryos at day 0, day 6 and day 12. The results showed that the CO2 concentrations in the treatment and control incubator were achieved the target levels of about (0.96±0.04)%, lower than 0.25%. There was no significant difference between the two groups in hatchability and fertilized egg hatchability, where were (89.17±2.53)% and (91.57±1.03)% in the treatment and control group, respectively. Moreover, the higher CO2 during early stage did not have much effect on the chick quality. The vascular development density of CAM on the 11th day had no significant difference between the treated and control groups, because the O2 concentration was maintained around (20.2±0.1)%. There was also no significant difference in the calcium content of eggshells and embryos in the treated and control groups (P>0.05), but the calcium content of embryos increased significantly from day 6 to day 12 in both groups (P<0.05). However, the embryo weight and relative embryo weight in the treatment group were significantly higher than those in the control group on the 9th and 12th day (P<0.05), but they were not consistent on day 6, day 15, and day 18 (P>0.05). The effect of higher CO2 on the embryo weight only occurred around the final stage of CO2 stimulation. The reason was that the liquefaction of protein by CO2 contributed greatly to promote the embryo absorption of nutrients from the protein, thereby accelerating embryonic development. But the effects just remained for a short term, until the CO2 level was lower. Furthermore, the albumen pH in the treatment group was lower than that in the control group, due mainly to the higher external CO2 level, particularly the significant differences on the 3rd, 6th, and 9th days. The overall change trend of protein pH value increased first and then decreased, finally reaching the peak on day 3. Consequently, 1% CO2 treatment during the early stage of incubation (0-10 d) lowered the protein pH, while accelerated the embryo development without affecting the hatchability and chick quality.