Fish is an important source of food and income for a significant portion of the Amazonian population, especially those who live along the rivers and lakes in the region. Pseudoplatystoma punctifer (Castelnau, 1855), known as surubim, is a species of Neotropical catfish widely exploited by fisheries and commercially valuable in the Guaporé River basin, Brazil. However, population dynamics are poorly known in the region. To understand the population structure and reproductive biology aspects of the surubim, monthly experimental fisheries were carried out in the São Miguel River, state of Rondônia, Brazil, between August 2020 and July 2021. The captured fish were weighed, and its gonads were removed and weighed for histological analyses (microscopic description) and classification of the maturation stages (macroscopic description). The collected gonads were fixed in 10% buffered formalin, dehydrated, cut into 4‐μm‐thick sections, and stained with hematoxylin and eosin. A total of 34 individuals were collected (20 females and 14 males), showing positive allometric growth and condition factor from 0.81 to 1.79 for females and males, respectively. Females were larger in size and weight than males. P. punctifer reaches its first maturation at an average length of 68.30 cm, indicating the importance of respecting the minimum capture size as an alternative to ensure the natural stocks of this fishery resource in the region. The results provide support for the establishment of public policies and actions for conservation, management, and regulation of fishing.