Anxiety disorders significantly affect females. Alterations in the production and release of melatonin may trigger pathology-related symptoms. We evaluated the effect of pinealectomy on the estrous cycle and anxiety-like behavior in female rats. The study was approved by the Animal Research Ethics Committee of the Federal University of Sergipe (CEUA/UFS), No. 4809100424. A total of 43 Wistar rats were used, divided into: control (CTR), sham (SHAM), and pinealectomized (PIN). The animals underwent pinealectomy at 30 days of age. Behavioral tests were conducted to assess anxiety-like and motor behavior at two, four, and six months of age. The estrous cycle was monitored throughout the experiment. Pinealectomy did not cause motor impairment in the animals, as shown by the total distance traveled in the open field. Evaluation of the time spent in the center zone revealed an increase in anxiety-like behavior in the PIN group compared to CTR and SHAM, evidenced by the reduced time spent in this zone. In the elevated plus maze test, anxiety-like behavior alterations were increased in the PIN group compared to CTR and SHAM, due to a decrease in time spent in the open arm and an increase in time spent in the closed arm, as well as a reduction in the number and duration of head dips in the perforated plate. No effect of the estrous cycle was observed. The findings indicate that pinealectomy is associated with an increase in anxiety-like behavior, regardless of the estrous cycle.