Background: Despite recent advances with newer androgen receptor(AR) pathway inhibitors, treatment resistance in castrate resistant prostate cancer continues to remain a clinical problem. Co-targeting approaches are of significant interest to slow the progression of disease and delay the onset of resistance. With both Akt and MEK pathways becoming activated as prostate cancer develops resistance to AR-targeted therapy, this study explores co-targeting these pathways in AR positive prostate cancer models. Methods: Using in vitro models of prostate cancer progression from androgen dependent to castrate resistant and ENZ-resistant disease, we evaluated the effect of Akt and/or MEK inhibition with AZD5363 and PD0325901, respectively, on cell proliferation, apoptosis and downstream signalling pathways. For in vivo models, we tested the combination of Akt and MEK inhibition castrated mice bearing MR49F and 22RV1 cells which are resistant to ENZ. Results: Inhibition of Akt reduced S6 phosphorylation, inhibited growth and induced apoptosis in LNCaP-derived cells; the addition of a MEK inhibitor increased apoptosis and cell cycle arrest, but did not further decrease cell proliferation. In contrast, PTEN wild-type 22RV1 cells demonstrated a greater sensitivity to MEK inhibition, but were generally insensitive to Akt inhibition. In vivo, combination of Akt and MEK inhibition resulted in more consistent tumour growth inhibition of MR49F xenografts and longer disease specific survival than Akt inhibitor monotherapy, with induction of pERK staining being evident in some AZD5363 monotherapy treated tumours. Conversely, 22RV1 xenografts had greater resistance to Akt inhibition and greater sensitivity to MEK inhibition. Conclusions: Our data suggest that combination of Akt and MEK inhibition may improve prostate cancer control in ENZ-resistant prostate cancer. Further, with the major effect in both MR49F and 22RV1 xenografts attributable to one inhibitor, our results also suggest the importance of characterizing the dominant oncogenic pathway in each patient’s tumour in order to select optimal therapy.