Organochlorine pesticides (OCs) have been associated with breast cancer development and progression. However, the deleterious mechanisms exerted by these contaminants are yet unclear and need to be further elucidated. In the present study, we investigated the effects of a number of OCs (previously detected in human serum from a Spanish population), individually or in combination, on normal human mammary epithelial cells (HMEC) at concentrations close to those found in human beings. The results obtained after a 96-h exposure indicated that OCs exert a clear cytotoxic effect on these cells at higher concentrations than those found in human beings. DDT-derivative organochlorines (DDT and its metabolites, DDE and DDD) are individually more cytotoxic than non-DDT-derivative organochlorines (aldrin and dieldrin). On the contrary, combinations of non-DDT organochlorines were clearly more cytotoxic than combinations of DDT-derivative organochlorines at concentrations close to those described in human serum. Additionally, transcriptional regulation arrays showed that the exposure of HMEC to an environmentally relevant mixture of OCs (p,p'-DDD plus p,p'-DDE plus o,p'-DDE plus aldrin plus dieldrin) sharply upregulated the expression of a number of protein kinases genes, such as ACVRL1, ALK-1, KIT, ERBB3, and ALK-1 at concentrations close to those detected in human populations. Taken together, these findings show a detrimental effect of OCs on human breast cells and indicate a possible association between exposure to organochlorine pesticide combinations and the induction of transformation processes in human breast cells.