To inform the conservation of the Regionally Critically Endangered West African lion, we studied lion population size and social structure in Niokolo Koba National Park (NKNP), Senegal. Calling station and camera trap surveys were conducted and opportunistic lion observations were documented from 2015 to 2020. The average lion density was calculated as 0.50 lion/100 km² and the population size was estimated as 28–56 individuals. Average lion group size was 2.1 ± 1.45 lions (range 1–8 lions). There were no significant differences between observations of single individuals (43%), groups of two (22%), three (19%), four (4%) and more than four (5%) individual lions (X² = 30.021, p-value = 0.06452). Sex ratio showed a ratio of male:female of 1:1.1 to 1:2.8. About 13.59% of the lion population was composed of cubs while, respectively, 81.22% and 5.17% were adults and subadults. Based on the analysis of activity patterns, lions in NKNP are mainly nocturno-crepuscular (18:00–01:00) and matutinal (06:00–08:00). Lion population size and density are still low in NKNP compared to earlier estimates from the same area, in the late 1990s. We therefore conclude that the conservation of lions in NKNP must be further improved to safeguard this population in the long term.