Background: Anopheles darlingi is the main malaria vector in the Amazon region and is among the most efficient malaria vectors worldwide. However, due to the lack of a well-established laboratory colony, key control-relevant aspects of the bionomics, behaviour, genetics, and vector-parasite relationships of An. darlingi remain unknown. Here, biological parameters that had been successful in initiating other Anopheles colonies were optimized and improved for An. darlingi, with the aim of establish a free-mating, stable, and highly productive laboratory colony. Methods: Wild An. darlingi adult females were field collected from Zungarococha, Loreto Department, Peru (03 °49′32.40″S, 73° 21′00.08″W), and taken to the NAMRU-6 Insectary in Iquitos where F<inf>1</inf> offspring were produced and reared. Natural copulation was successfully induced in F<inf>1</inf> adults under a thermoperiod of 30 ± 1 °C during the day and 25 ± 1 °C at night, and with a 30-min LED light stimulation period at dusk. Oviposition success was enhanced using egg-laying containers with a dark-coloured surface. Larval feeding regimes were standardized for optimal larval development. Optimized copulation induction methods were used to facilitate mating in An. darlingi until the F<inf>10</inf> generation. No copulation induction assistance was needed in subsequent generations. Results: In 19 generations, the An. darlingi colony produced a total of 763,775 eggs; 441,124 larvae; 248,041 pupae; and 231,591 adults. A mean of 0.56 sexual encounters/female/cage (n = 36 cages) was recorded across the first ten generations (F<inf>1</inf>-F<inf>10</inf>). A mean insemination rate of 54.7 % (n = 5,907 females) ranging from 43.6 % (F<inf>2</inf>) to 66.6 % (F<inf>10</inf>) was recorded across nine generations (F<inf>2</inf>-F<inf>10</inf>). Free-mating was casually observed in the F<inf>8</inf> generation, and subsequently confirmed in the F<inf>9</inf> and F<inf>10</inf> generations; comparable insemination rates and egg laying between stimulated (51.6 %, 12.9 eggs/female), and non-stimulated (52.3 %, 11.2 eggs/female) females were recorded. The time from egg to adult development ranged from 10 to 20 days. Moreover, the colony was relocated to a new laboratory within Iquitos in the F<inf>14</inf> generation without any noted changes in its productivity. By March 2015, the An. darlingi colony has been successfully reared to the F<inf>26</inf> generation. Conclusions: This constitutes the first report of a free-mating, highly productive, and long-standing An. darlingi laboratory colony established through natural copulation induction, which will support critical malaria research. This rearing methodology may be a transferable, cost-effective alternative to labour-intensive forced mating practices widely used in maintaining other Anopheles colonies.