Tooth loss constitutes a major public health challenge, sharing common risk factors with non-communicable diseases. To report the relationship between tooth loss and serum cotinine levels in a population sample of mixed ethnic heritage from the Belville South area in South Africa. Cross-sectional epidemiological study.Subjects were invited from 2014 to 2016 according to a consecutive sampling technique and all those who met the inclusion criteria were included. In all, 1876 individuals were included, being 1416 females (75.5%), with a combined average age of 49.5 ± 15.3 years. In total 46.7% of the sample was edentulous, with females presenting a higher proportion than males (50.7% vs. 34.1%, p < 0.001). The relative risk (RR) of being edentulous was higher for females (RR=1.8, 95% CI=1.35-2.41, p<0.001) and for participants with cotinine levels 15-299 ng/ml (RR = 1.37, 95% CI=1.02=1.83, p=0.04) and ≥300 ng/ml (RR=1.51, 95% CI=1.09-2.08, p=0.01). Maxillary incisors and mandibular molars were the most prevalent missing teeth. The burden of tooth loss is high in the studied population sample, as well their unmet needs for dental care. Female gender, tobacco exposure, and aging were associated with partial and total edentulism.