This study was undertaken to examine the effect of GH treatment during a pause in laying on (1) ovarian follicle formation, growth (folliculogenesis), and atresia; (2) follicle cell proliferation and apoptosis; and (3) mRNA expression of selected yolk-specific proteins in the chicken liver. A pause in egg laying was induced by food deprivation for 5 d, followed by feeding every other day, and then feeding daily from day 10 onward. Birds were divided into 3 groups: control (n = 18) fed ad libitum, subjected to a pause in laying (n = 18), and subjected to a pause in laying and injected every day with 200 μg/kg BW of chicken GH (chGH; n = 18). The liver, ovarian stroma, and follicles were isolated from the hens of each group on days 6 (ovary regression), 13 (ovary recrudescence), and 17 or 20 (ovary rejuvenated) of the experiment. The results showed that injection of chGH during fasting (1) increased the number of follicles <1 mm and proliferating cell nuclear antigen (PCNA)-positive (proliferating) cells in these follicles; (2) attenuated the expression of PCNA and survivin mRNA in the white follicles and the activity of caspases 3, 8, and 9 in the stroma and white follicles; (3) intensified the atresia of yellow hierarchical follicles; and (4) deepened the effect of starvation on egg yolk gene expression concomitantly with considerably increased IGF-1 transcription levels in the liver (P < 0.05 to P < 0.001). Prolongation of chGH injections into the refeeding period did not exert pronounced effects on the examined parameters. In summary, the results provide evidence that GH promotes the formation and development of prehierarchical follicles in the hen ovary during a pause in laying by regulating cell proliferation and apoptosis. Alterations in cell proliferation- and apoptosis-related gene expression or enzyme activity in ovarian follicles as well as the expression of egg yolk proteins in the liver after chGH treatment strongly suggest that this hormone is involved in determining the rate of regression and rejuvenation of the chicken ovary during a pause in laying.