Background and Purpose Dopamine agonists targeting D-2 receptor have been used for decades in treating pituitary adenomas. There has been little clear evidence implicating the canonical G protein signalling as the mechanism by which D-2 receptor suppresses the growth of pituitary tumours. We hypothesize that beta-arrestin2-dependent signalling is the molecular mechanism dictating D-2 receptor inhibitory effects on pituitary tumour growth. Experimental Approach The involvement of G protein and beta-arrestin2 in bromocriptine-mediated growth suppression in rat MMQ and GH3 tumour cells was assessed. The anti-growth effect of a beta-arrestin2-biased agonist, UNC9994, was tested in cultured cells, tumour-bearing nude mice and primary cultured human pituitary adenomas. The effect of G protein signalling on tumour growth was also analysed by using a G protein-biased agonist, MLS1547, and a G beta gamma inhibitor, gallein, in vitro. Key Results beta-arrestin2 signalling but not G protein pathways mediated the suppressive effect of bromocriptine on pituitary tumour growth. UNC9994 inhibited pituitary tumour cell growth in vitro and in vivo. The suppressive function of UNC9994 was obtained by inducing intracellular reactive oxygen species generation through downregulating mitochondrial complex I subunit NDUFA1. The effects of G alpha i/o signalling and G beta gamma signalling via D-2 receptor on pituitary tumour growth were cell-type-dependent. Conclusion and Implications Given the very low expression of G alpha i/o proteins in pituitary tumours and the complexity of the responses of pituitary tumours to G protein signalling pathways, our study reveals D-2 receptor beta-arrestin2-biased ligand may be a more promising choice to treat pituitary tumours with improved therapeutic selectivity.