The effectiveness of ex situ breeding programs for endangered species can be limited by challenges in mimicking mating competitions that naturally occur among multiple mates in the wild. The objective of this study was to evaluate the impact of timed natural matings and/or artificial inseminations in the context of the urinary estrogen surge on cub production in the giant panda (Ailuropoda melanoleuca). We used a large cohort of giant pandas, including 12 females and 17 males. DNA paternity exclusion was used to pinpoint accurately the interval during the estrogen surge that coincided with the ideal sperm deposition time to produce offspring. Of the 31 cubs (in 19 pregnancies), 22 (71.0%; 15 pregnancies) were produced from matings occurring on the day of or the day after the maximal urinary estrogen peak. Sixteen of the 19 pregnancies (84.2%) produced at least one offspring sired by the first male mating with the dam. There was a preponderance of twins (12 of 19; 63.2%), and dual paternities were discovered in 3 of 12 twin sets (25%). These findings indicate a strong relationship between the excreted estrogen surge and sperm deposition to achieve pregnancy in the giant panda. To ensure the production of the most genetically diverse young, it is imperative that the most appropriate male mate first and on the day of or the day after the highest detected estrogen value. There is no advantage to increasing the number of copulations or mating partners within 1 day of the estrogen peak on the incidence of twinning, although this practice may increase the prevalence of dual paternity in cases of multiple births.