The development of safer mu opioid receptor (MOR) agonists with reduced side effects is a key focus of pain research. Some studies have suggested that MOR agonists with reduced β-arrestin 2 (βArr2) signaling (i.e. G-protein biased agonists) may have greater therapeutic windows. However, there have been a several conflicting reports, and it is not clear what role, if any, βArr2 signaling plays in MOR-mediated analgesia, tolerance, or side effects. Therefore, we used βArr2 knockout mice to systematically investigate the causal role of βArr2 signaling in antinociception, antinociceptive tolerance, respiratory depression, constipation, and reward induced by morphine and the two novel MOR agonists, kurkinorin and kurkinol. Kurkinorin and kurkinol exhibited potent antinociceptive effects that were reversed by MOR knockout. Unlike morphine or kurkinorin, our most G-protein biased agonist, kurkinol, showed no significant tolerance after seven days of ~2×ED dosing. However, in a chemotherapy-induced neuropathic pain model, all three compounds were ineffective after 20 days of ~ED dosing, indicative of tolerance. All compounds exhibited significant MOR-dependent side effects, though kurkinorin had reduced gastrointestinal and respiratory depressive effects compared to morphine despite exhibiting less G-protein bias. Knockout of βArr2 significantly increased antinociceptive potency for morphine and kurkinorin but not kurkinol, and otherwise had no significant impact on tolerance or any side effect tested. These results largely suggest that βArr2 signaling does not drive MOR-mediated antinociceptive tolerance, respiratory depression, constipation, or reward and do not support the development of G-protein biased compounds as a broadly effective strategy to reduce side effects.