Persistent pain can be managed with opioids, but their use is limited by the onset of tolerance. Ultramicronized N-palmitoylethanolamine (PEA) in vivo delays morphine tolerance with mechanisms that are still unclear. Since glial cells are involved in opioid tolerance and mast cells (MCs) are pivotal targets of PEA, we hypothesized that a potential mechanism by which PEA delays opioid tolerance might depend on the control of the crosstalk between these cells. Morphine treatment (30 μM, 30 min) significantly increased MC degranulation of RBL-2H3 cells, which was prevented by pre-treatment with PEA (100 μM, 18 h), as evaluated by β-hexosaminidase assay and histamine quantification. The impact of RBL-2H3 secretome on glial cells was studied. Six-hour incubation of astrocytes with control RBL-2H3-conditioned medium, and even more so co-incubation with morphine, enhanced CCL2, IL-1β, IL-6, Serpina3n, EAAT2 and GFAP mRNA levels. The response was significantly prevented by the secretome from PEA pre-treated RBL-2H3, except for GFAP, which was further upregulated, suggesting a selective modulation of glial signaling. In conclusion, ultramicronized PEA down-modulated both morphine-induced MC degranulation and the expression of inflammatory and pain-related genes from astrocytes challenged with RBL-2H3 medium, suggesting that PEA may delay morphine tolerance, regulating MC-astrocyte crosstalk.