The influence of dietary protein intake on muscle mass in adults remains unclear. Our objective was to investigate the association between protein intake and muscle mass in 31,278 men and 45,355 women from the Lifelines Cohort. Protein intake was estimated by food frequency questionnaire and muscle mass was estimated from 24 h urinary creatinine excretion. The age range was 18–91 years and mean total protein intake was 1.0 ± 0.3 g/kg/day. Across increasing quartiles of total protein intake, animal protein intake, and fish/meat/egg protein intake, creatinine excretion significantly increased in both men (+4% for total and +6% for fish/meat/egg protein intake, p < 0.001) and women (+3% for total and +6% for fish/meat/egg protein intake, p < 0.001). The associations were not systematically stronger or weaker with increasing age, but associations were strongest for young men (26–45 years) and older women (>75 years). The association between total protein intake and muscle mass was dependent on physical activity in women (p interaction < 0.001). This study suggests that total protein intake, animal protein intake, and in particular fish/meat/egg protein intake may be important for building and preserving muscle mass. Dietary protein sources should be further studied for their potential to build and preserve muscle mass.