Bicycling has multiple health benefits. Child-rearing may influence bicycling, but little is known about the impact of this between men's and women's bicycling, or of number and ages of children on bicycling. This study examined the longitudinal associations between having dependent children and bicycling for transportation and recreation over 4 years among mid-aged men and women. Data were from the HABITAT study (Australia). We analysed data from three survey waves (2007, 2009, 2011) using multilevel logistic regression stratified by gender (n = 7758). Findings indicate that having dependent children was associated with bicycling for transportation and recreation in contrasting ways for men and women. The odds of bicycling were higher in men with ≥2 children aged under 18y than men without children (transportation: OR = 1.93, 95% CI: 1.26, 2.98; recreation: OR = 2.37, 95% CI: 1.67, 3.37). Over time, the odds of recreational bicycling were lower in women with ≥2 children than women without children (OR = 0.83, 95% CI: 0.73, 0.93). However, for both men and women, the odds of recreational bicycling were higher in those with children aged 6–12y than those with younger or older children (men: OR = 1.86, 95% CI: 1.39, 2.49; women: OR = 1.79, 95% CI: 1.31, 2.46). Interventions to promote bicycling must therefore consider gendered differences in bicycling for travel and active leisure, and family circumstances. An opportunity to promote bicycling might be to target parents with children aged 6–12y.