The secretome of Mesenchymal Stromal Cells (MSCs) has demonstrated effectiveness in the treatment of Inflammatory Bowel Disease (IBD), offering a safer and more predictable alternative to the direct administration of MSCs. While parenteral administration is common, rectal delivery of the secretome provides targeted treatment for localized conditions such as ulcerative colitis and proctitis. Alginate-Laponite 3D-printed suppositories (Alg-Lap 3DPS) have shown a remarkable versatility in their ability to encapsulate and deliver therapeutic agents during previous studies. These formulations are also adaptable in the release of molecules with different physicochemical properties, and a Bovine Serum Albumin (BSA) displacement strategy has been proposed in order to promote their disintegration and controlled release. As such, these 3DPS formulations represent highly versatile pharmaceutical forms, enabling the rectal delivery of diverse molecules with controlled release characteristics. In this study, we combined the therapeutic potential of Adipose tissue derived-MSCs-secretome (AT-S) with the versatility and efficacy of Alg-Lap 3DPS. Herein, we demonstrate that these formulations are well-suited for the encapsulation and release of AT-S, confirming their ability to release bioactive factors from AT-S such as Galectin-9 upon applying the displacement strategy. Moreover, we validate the bioactivity of the released factors in vitro by testing their effects on pro-inflammatory M1-polarized macrophages. Notably, this study not only confirms the anti-inflammatory capacity of the AT-S-loaded 3DPS in vitro but also aims to elucidate the underlying mechanisms driving these immunomodulatory effects, revealing the inherent immunomodulatory capacity of the Alg-Lap matrices themselves in M1 macrophages and establishing the AT-S-loaded 3DPS formulations as innovative therapeutic approaches for the treatment of IBD.