This study assessed the polychlorinated biphenyl (PCB) levels in human milk and its associated health risk to infants from rural and urban settings of five districts of Punjab Province, Pakistan. The ∑34PCB concentrations ranged from 30.9 to 68.3 ng g-1 on lipid weight (l.w.) basis. The ∑8DL-PCB concentrations were ranged from 0.29 to 1.35 ng g-1 l.w., (mean 6.2 ± 8.7 ng g-1 l.w.), with toxicity equivalent to polychlorinated dibenzodioxins (PCDDs) ranging from 8.58 × 10-6 to 0.05 ng TEQ g-1 l.w. The spatial trend of PCB levels in human milk revealed higher bioaccumulative levels for urban mothers as compared with rural counterparts. The estimated daily intake (EDI) values of DL-PCBs to infants through trans-mammary transfer were considerably higher than tolerable daily intake limits established by WHO (i.e., 1-4 pg TEQ kg-1 bw) and other globally recognized organizations. Similarly, the hazard quotient values for TEQ ∑8DL-PCBs (range 1.21 to 79.87) were far above the benchmark value of 1 at all the sampling sites, indicating the high levels of adverse health risks to infants in the region through breast milk consumption. The ∑34PCB levels were found to be negatively correlated with mother' age (r = -0.31; p = 0.02), parity (r = - 0.85; p = 0.001), and infant' birth weight (r = - 0.73; p = 0.01). The present study suggests undertaking comprehensive public health risk assessment studies and firm regulatory measures to safeguard human health risks.