Background: Omega-3 polyunsaturated fatty acids, specifically the fish oil derived eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been proposed as inflammation resolving agents via their effects on adipose tissue. Objective: We proposed to determine the effects of EPA and DHA on human adipocyte differentiation and inflammatory activation in vitro. Methods: Primary human subcutaneous adipocytes from lean and obese subjects were treated with 100μM EPA and/or DHA throughout differentiation (differentiation studies) or for 72 hours post-differentiation (inflammatory studies). THP-1 monocytes were added to adipocyte wells for co-culture experiments. Subcutaneous and visceral adipose explants from obese subjects were treated for 72 hours with EPA and DHA. Oil-RedO staining was performed on live cells. Cells were collected for mRNA analysis by qPCR and media collected for protein quantification by ELISA. Results: Incubation with EPA and/or DHA attenuated inflammatory response to LPS and monocyte co-culture with reduction in post-LPS mRNA expression and protein levels of IL6, CCL2, and CX3CL1. Expression of inflammatory genes was also reduced in the endogenous inflammatory response in obese adipose. Both DHA and EPA reduced lipid droplet formation and lipogenic gene expression without alteration in expression of adipogenic genes or adiponectin secretion. Conclusions: EPA and DHA attenuate inflammatory activation of in vitro human adipocytes and reduce lipogenesis.