Insulin-like peptide 3 (INSL3) and sex steroids were measured in bovine dominant follicles and corpora lutea during the estrus cycle and in follicular cysts. Paired ovaries from beef heifers (n = 47) were classified, by their morphological features, either into four stages of the estrus cycle (Day 1 = day of ovulation, Day 20 = day of estrus) as Stage I (Days 1–4; n = 8), Stage II (Days 5–10; n = 10), Stage III (Days 11–17; n = 8), and Stage IV (Days 18–20; n = 11) or follicular cystic (n = 10). Cysts (n = 15) were subdivided into estrogen-active (n = 7) and estrogen-inactive (n = 8) cysts. INSL3, testosterone, and estradiol-17β concentrations in the dominant follicular fluid of Stage IV were higher than those in Stages II and III (P < 0.05). INSL3 concentrations in the cystic fluid were similar to those in dominant follicles at Stage IV, whereas testosterone and estradiol-17β concentrations were lower in cysts (P < 0.05). INSL3 content per estrogen-inactive cyst was higher than that of Stage IV (P < 0.05). INSL3 and progesterone concentrations in luteal tissue and contents per corpus luteum were higher in Stages II and III (P < 0.05). In conclusion, INSL3 secretion in bovine dominant follicles increased with maturation. Follicular cysts may retain the production of INSL3 during their formation but tend to lose the capacity for testosterone secretion. Estrogen-inactive cysts subjected to advanced atresia may accumulate more INSL3. INSL3 production in bovine corpora lutea is enhanced during maturation.