We investigated if prenatal exposures to tobacco smoke lead to changes in mitochondrial DNA content (mtDNA) in cord serum and adversely affect newborns’ health. Umbilical cord serum cotinine levels were used to determine in utero exposure to smoking. Cord serum mtDNA was measured by quantitative polymerase chain reaction analysis of the genes coding for cytochrome c oxidase1 (MT-CO1) and cytochrome c oxidase2 (MT-CO2). Log transformed levels of mtDNA coding forMT-CO1andMT-CO2were significantly higher among infants of active smokers with higher serum level of cotinine (p < 0.05) and inversely associated with gestational age (p = 0.08;p = 0.02). Structural equation modeling results confirmed a positive association between cotinine andMT-CO1and2 (p < 0.01) and inverse associations with gestational age (p = 0.02) andIGF-1(p < 0.01). We identified a dose-dependent increase in the level ofMT-CO1andMT-CO2associated to increased cord serum cotinine and decreased gestational age.