Berberine (BBR) is a nitrogenous cyclic natural alkaloid with potential anticancer activity. However it has been less explored due to its poor pharmacokinetic profile. Dendrimers (e.g. PAMAM) have promising potential to deliver anticancer drugs/bio-actives because of their well-defined architecture, monodispersity and tailor-made surface functionality. In the present study it was attempted to deliver berberine through G4 PAMAM dendrimers by conjugation (BPC) as well as encapsulation (BPE) approach. The developed encapsulated and conjugated berberine formulations were found to have size in the approximate range of 100–200 nm while zeta potential was almost same as PAMAM G4 dendrimer. The entrapment efficiency in BPE was found to be 29.9%, whereas, the percentage conjugation in BPC was found to be 37.49% indicating high drug payload in conjugation. The developed nano-formulations were characterized through 1H NMR, FT-IR as well as electron microscopy (SEM and TEM). The in vitro release study in different media (water and PBS 7.4) showed sustained release pattern of BBR. Almost 72% and 98% drug was released within 24 h respectively; whereas in PBS almost 80% and 98% release was observed within 24 h, respectively. The formulations followed Higuchi release and first order release as best fit release kinetic model. MTT assay results showed significantly higher anticancer activity for the PAMAM-BBR (BPC) (p < 0.01) against MCF-7 and MDA-MB-468 breast cancer cells. The time dependent ex vivo hemolytic toxicity of the BPC and BPE was significantly less (<5%) even after 24 h, which indicated that the formulations can be regarded as significantly safe and biocompatible. Similarly, the in vivo hematological parameters were analyzed through auto-analyzer and the formulations were found to be safer and biocompatible with very least but insignificant (p > 0.05) effects. The in vivo pharmacokinetic parameters were found to be impressively improved in albino rat model. The pharmacokinetic parameters such as half-life (t1/2) and AUC of berberine were impressively improved in the plasma level time in vivo studies in albino rat model. The obtained t1/2 was 14.33 h for BPC compared to 6.7 h for BBR alone. The overall conclusion says that among both the developed formulations the conjugated formulation (BPC) was found to be more prominent than the encapsulated one (BPE). Therefore conclusively conjugation can be a better option for the delivery of natural bio-actives through dendrimers.