The objectives of the present study were to evaluate the effect of increased milking frequency (IMF) during early lactation on milk yield, proliferation and apoptosis in mammary epithelial cells of F₁ Holstein × Zebu cows. Fourteen cows F₁ Holstein × Zebu cows were randomly distributed into 2 groups. The control group (2×) was milked twice a day up to 210 d in milking (DIM), and the group subjected to IMF (4×) was milked 4 times a day from 2 to 21 DIM, and twice a day from 22 to 210 DIM. Milk production was measured daily from 4 to 30 DIM and then each 15 d until 210 d. Mammary biopsies were performed on d 2, 7, 14, 21, and 28 postpartum. Biopsies were obtained using a biopsy needle (12 g × 12). Mammary tissue was used to measure rates of cell proliferation and apoptosis using Ki-67 nuclear proliferation antigen localization and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL), respectively. Data were analyzed with a repeated measures design and means were compared by Tukey's test (P < 0.05). During IMF, the 4× produced 2.6 ± 0.2 kg/d more milk than the 2× (P < 0.05). After IMF phase, milk production from the 4× decreased and was similar to 2× up to 210 DIM (P > 0.05). Mammary cell proliferation and apoptosis were not affected by milking frequency (P > 0.05). Rates of proliferation and apoptosis were influenced by days of lactation (P < 0.05). On the second day of lactation, the highest apoptosis rate (0.3%) was observed. Days 7 (0.16%), 14 (0.13%), 21 (0.11%) and 28 (0.06%) had similar and lower rates in comparison with the second day of lactation. There was a higher percentage of proliferation in mammary epithelial cells on d 2 (2.12%). On d 7 (0.99%) and 14 (0.87%), the proliferation rate dropped to about half the value observed on d 2 and in the following week (0.47%) it was observed another reduction which remained until the 28th of lactation (0.43%). Changes in milking frequency during early lactation did not alter milk yield after IMF and mammary cell population dynamics suggesting that maybe the mammary biopsy procedure used in the current study influenced these responses negatively.